MICROBIOLOGY REVIEWER INSTRUMENTS 1.
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– set at 35-37’C 35- 37’C INCUBATOR – set Quality Control: Monitoring of temperature at 35’C for MRSA, not at 37’C. Also for viral culture (37’C) 18-24 hrs (aerobic culture) 24-48 hrs (anaerobic culture) DURHAM TUBE For water bacteriology Gas detector INOCULATING NEEDLES (<5cm) Bent Wireloop: Wireloop: Used for Fungal Culture Calibrated Wireloop: a. For quantitative technique: important in colony count in urine samples b. Diameter Size: 2mm (0.001 ml urine = 1,000 loop factors) c. 70% Ethyl Alcohol – Alcohol – used used as a disinfectant, better than using fire for disinfection d. 70% Ethyl Alcohol with SAND – used – used for SPUTUM specimen, used to dislodge the stickiness of the sputum since flame can cause aerosol formation when heated. Nichrome Inoculating Needles – – contains iron, can cause false positive in oxidase test. Use an applicator stick instead of inoculating needle for oxidase test. COTTON SWAB Only small amount of organism is obtained (carrier state) Toxic to Neisseria Charcoal is added in culture media to remove the toxicity of cotton 2 Swabs needed for: st 1. Culture (1 ) nd 2. Gram Stain (2 ) TYPING SERA For Salmonella-Shigella Detects Antigen a. O – Somatic – Somatic b. H – Flagellar – Flagellar – used for Mantoux Test or Purified Protein Derivative TUBERCULIN SYRINGE – (PPD): a method for the skin test for Tuberculosis – transfer liquid PASTEUR PIPETTE – transfer
HISTORY 1.
Anton Van Leeuwenhoek First to describe the bacteria
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Robert Koch Germ Theory “It is the organism that causes human disease” First to isolate bacteria (Pure Culture) NOTE: Culture is a definitive test/gold standard Louis Pasteur – Pasteur – Father Father of Modern Micro Ehrlich – Ehrlich – First First to use dyes for stain
CHARACTERISTIC OF BACTERIA 1.
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Prokaryotic No nuclear membrane, no mitochondria, small than Eukaryotic Fungi is Eukaryotic (same as human) Virus is NEITHER prokaryotic nor eukaryotic Has both DNA and RNA Multiply by Binary Fission Measured in Micrometer (um) Cell wall (except Mycoplasma) Main composition: Peptidoglycan Mycoplasma is the smallest bacteria o first bacteria to be cloned o no cell wall, reason why it is resistant to antibiotic like Penicillin, o and not gram stained Incubated aerobically (CO2, CAP) o Classification Phenotype – Phenotype – Observable Observable Genotype – Genotype – DNA DNA type (PCR) 0.4 – 2um 2um Size: 0.4 –
PARTS OF BACTERIA 1.
– aka. Slimy Layer CAPSULE – aka. Mucoid colony in culture Virulence Factor and Anti-phagocytic OPSONIN (IgG) – – antibody that facilitates phagocytosis of o encapsulated bacteria Capsular Antigen (K Ag or Vi Ag) Vi Ag = Salmonella typhi, causative agent for Typhoid Fever ( Mary , o the first person to spread typhoid fever) Neufeld Quellung Test – Test – serologic serologic test for capsular antigen o Reagent : Anti-Sera (Antibody against capsular antigen)
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Bacteria commonly used: a. Streptococcus pneumoniae b. Neisseria meningitides c. H. influenzae (Serotyping) – (Serotyping) – used used for identification and vaccine making
CELL WALL Responsible for bacterial morphology Gives shape to bacteria Basis of gram stain Gram (+): PURPLE o Thick Peptidoglycan (reason why it cannot be decolorized by alcohol/insoluble) Teichoic Acid Gram (-): RED o LPS Thin Peptidoglycan (soluble, easier to be decolorized) Periplasm Note: Since MYCOPLASMA doesn’t have cell wall, its shape is PLEOMORPHIC, can be bacilli, coccobacilli, etc. ENDOTOXIN – ENDOTOXIN – found found at LPS EXOTOXIN found at gram (+), is more dangerous than Endotoxin since Exotoxin is focused one site, while ENDO is systemic. Botulinum Toxin – Toxin – most most potent toxin to human
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PLASMA MEMBRANE Site for energy synthesis Osmotic or permeability barrier Hypertonic/Hypotonic solution cannot be destroyed by bacteria o Regulate transport of nutrients in and out of the cell
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NUCLEOID 2 Types of DNA: 1. CHROMOSOME: DS-DNA 2. PLASMID: Extrachromosomal DNA Drug Resistance: 1. Chromosome Mediated Drug Resistance – MRSA – MRSA 2. Plasmid Mediated Drug Resistance – – ESBL (+) Organisms: (Gram Negative Rods) - more dangerous since it’s a transfer DNA Transfer DNA: “PLASMID” Transfer Gene: “TRANSPOSON” (Transfer ng Puson hahaha)
Gene Transfer of Bacteria: (For Gram Neg Bacilli) 1. Conjugation Plasmid mediated Sex Pili required 2. Transduction – Transduction – bacteriophage bacteriophage mediated (virus infecting bacteria) Ex. Corynebacterium diptheriae – – gene is carried upon by a bacteriophage 3.
Transformation – Transformation – naked naked DNA (virulent avirulent Ex. Streptococcus pneumoniae
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METACHROMATIC GRANULES Cannot be seen in gram stain, special stain is needed (such as Methylene Blue) Food reserves (Corynebacterium) – Babes – Babes Ernst/Volutin Granules
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RIBOSOMES For protein synthesis; 70S for Bacteria, 80S for Fungi
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PILI OR FIMBRIAE (For Gram Negative) a. Common Pili – Pili – bacterial bacterial adherence attaches on the epithelium attracting phagocytes (PMNs for Neisseria gonorrhea PUS *Tulo) b. Sex Pili – Pili – gene gene transfer
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ENDOSPORES Calcium dipicolinate or dipicolinic acid For RESISTANCE; Endospores of Fungi is for Reproduction Bacillus, Clostrdium Autoclave is the best sterilization method because it is sporicidal killing the spores (Bacillus (Bacillus is is used as a control for sterilization) Iodine = Sporicidal; Alcohol = Non-Sporicidal; Soap = Germicidal
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FLAGELLA a. Monotrichous – Monotrichous – 1 1 polar flagella ( Vibrio, Pseudomonas aeruginosa) b. Amphitrichous – Amphitrichous – 2 2 polar flagella ( Spirillum minor) c. Lophotrichous – – group or tufts flagella ( Burkholderia, Stenotrophomonas) d. Peritrichous – – flagella around, MOST COMMON ( E. coli, enterobacteriaciae) H Antigen (Flagellar Ag) – used – used for motility test
10. AXIAL FILAMENTS (For Spirochetes)
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Bacteria commonly used: a. Streptococcus pneumoniae b. Neisseria meningitides c. H. influenzae (Serotyping) – (Serotyping) – used used for identification and vaccine making
CELL WALL Responsible for bacterial morphology Gives shape to bacteria Basis of gram stain Gram (+): PURPLE o Thick Peptidoglycan (reason why it cannot be decolorized by alcohol/insoluble) Teichoic Acid Gram (-): RED o LPS Thin Peptidoglycan (soluble, easier to be decolorized) Periplasm Note: Since MYCOPLASMA doesn’t have cell wall, its shape is PLEOMORPHIC, can be bacilli, coccobacilli, etc. ENDOTOXIN – ENDOTOXIN – found found at LPS EXOTOXIN found at gram (+), is more dangerous than Endotoxin since Exotoxin is focused one site, while ENDO is systemic. Botulinum Toxin – Toxin – most most potent toxin to human
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PLASMA MEMBRANE Site for energy synthesis Osmotic or permeability barrier Hypertonic/Hypotonic solution cannot be destroyed by bacteria o Regulate transport of nutrients in and out of the cell
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NUCLEOID 2 Types of DNA: 1. CHROMOSOME: DS-DNA 2. PLASMID: Extrachromosomal DNA Drug Resistance: 1. Chromosome Mediated Drug Resistance – MRSA – MRSA 2. Plasmid Mediated Drug Resistance – – ESBL (+) Organisms: (Gram Negative Rods) - more dangerous since it’s a transfer DNA Transfer DNA: “PLASMID” Transfer Gene: “TRANSPOSON” (Transfer ng Puson hahaha)
Gene Transfer of Bacteria: (For Gram Neg Bacilli) 1. Conjugation Plasmid mediated Sex Pili required 2. Transduction – Transduction – bacteriophage bacteriophage mediated (virus infecting bacteria) Ex. Corynebacterium diptheriae – – gene is carried upon by a bacteriophage 3.
Transformation – Transformation – naked naked DNA (virulent avirulent Ex. Streptococcus pneumoniae
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METACHROMATIC GRANULES Cannot be seen in gram stain, special stain is needed (such as Methylene Blue) Food reserves (Corynebacterium) – Babes – Babes Ernst/Volutin Granules
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RIBOSOMES For protein synthesis; 70S for Bacteria, 80S for Fungi
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PILI OR FIMBRIAE (For Gram Negative) a. Common Pili – Pili – bacterial bacterial adherence attaches on the epithelium attracting phagocytes (PMNs for Neisseria gonorrhea PUS *Tulo) b. Sex Pili – Pili – gene gene transfer
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ENDOSPORES Calcium dipicolinate or dipicolinic acid For RESISTANCE; Endospores of Fungi is for Reproduction Bacillus, Clostrdium Autoclave is the best sterilization method because it is sporicidal killing the spores (Bacillus (Bacillus is is used as a control for sterilization) Iodine = Sporicidal; Alcohol = Non-Sporicidal; Soap = Germicidal
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FLAGELLA a. Monotrichous – Monotrichous – 1 1 polar flagella ( Vibrio, Pseudomonas aeruginosa) b. Amphitrichous – Amphitrichous – 2 2 polar flagella ( Spirillum minor) c. Lophotrichous – – group or tufts flagella ( Burkholderia, Stenotrophomonas) d. Peritrichous – – flagella around, MOST COMMON ( E. coli, enterobacteriaciae) H Antigen (Flagellar Ag) – used – used for motility test
10. AXIAL FILAMENTS (For Spirochetes)
BACTERIAL PHYSIOLOGY 1.
Oxygen Requirement: a. Obligate Aerobe = With oxygen Ex. MYCOBACTERIA b. Obligate Anaerobe = Without oxygen Ex. BACTEROIDES FRAGILIS (normal flora c. Facultative Anaerobe = With or without oxygen Ex. PATHOGENS (most pathogenic bacteria are incubated AEROBICALLY) d. Microaerophiles = Low Oxygen (5% O 2 + 10% CO 2 + 85% N 2) Ex. CAMPYLOBACTER - Materials providing CO 2: Gas Pack, Candle Jar (3% CO 2), Campy Gas e. Aerotolerant Anaerobes = Not destroyed by O2, anaerobic but can tolerate oxygen Ex. LACTOBACILLUS
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Temperature: a. Psychrophilic = 0- 20’C (refrigerator) Ex. LISTERIA M. and YERSINIA E. Listeria – Listeria – agent agent of food poisoning Food contaminated with Listeria (mostly found in ref) 1. Coleslaw 2. Milk (Mam: “Ano ang gatas? Milk yan!”) and Cheese Yersinia e. e. – blood – blood bank contaminant (presence of bubbles in the blood bag) b. Mesophilic = 20-40’C 20-40’C (Pathogenic) Optimal pH (37’C) c. Thermophilic = 40- 60’C Ex. THERMUS AQUATICUS – AQUATICUS – source source of DNA in PCR Polymerase
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pH Requirement: a. Acidophilic Ex. LACTOBACILLUS Produces lactic acid Normal flora of GIT and Vagina Increased in: Pregnancy (protection against UTI) Promotes Candidiasis Candidiasis (fungi are also acidophilic) but inhibits Gardnerella vaginalis (vaginosis) vaginalis (vaginosis) since it is alkaline b. Neutrophilic – Pathogenic – Pathogenic Bacteria Ex. E. COLI c. Basophilic – Basophilic – VIBRIO VIBRIO
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Salt Concentration - Halophilic Ex. S. AUREUS = 7.5% NaCl ENTEROCOCCUS = 6.5% NaCl
NOTE: In growth culture: AEROBIC BACTERIA grows at the SURFACE; ANAEROBIC BACTERIA grows at the BOTTOM; MICROAEROPHILES grows at the MIDDLE; FACULTATIVE/AEROTOLERANT grows EITHER/ANYWHERE. Case Analysis: Analysis : In the case of bronchial washing, an organism grows on slide (Gram Stain (+)) but – ) (No growth). Culture ( – Remember that if Gram Stain (+), should be Culture (+)! Rationale: Bronchial washing is an aerobic sample. The bacteria is possibly anaerobic, and the bronchial washing might be exposed to oxygen already, the reason why there is no growth in the agar. Bronchial aspirate should be the specimen.
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Nutritional Requirement: a. Autotrophs/Lithotrophs = inorganic compound as carbon source. Ex. CO2 b. Heterotrophs/Organotrophs = organic compound as carbon source = mostly pathogenic bacteria Ex. Glucose
BACTERIAL METABOLISM 1.
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Respiration (Aerobic Process) a. Kreb’s cycle – aerobic – aerobic process b. Electron Transport Chain – Chain – aerobic aerobic process c. Glucose CO2 and H2O Oxidation (Aerobic Process) a. Glucose Acid Ex. NFO (Non Fermentative Organism) such as PSEUDOMONAS oxidizes sugar to produce acid. Fermentation (Anaerobic Process) a. Glycolysis (Embden Meyerhoff Pathway) b. Glucose Acid/Alcohol
NOTE: Oxidation and Fermentation BOTH produces ACID, but differ in Aerobic and
BACTERIAL GROWTH CURVE 1.
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LAG Phase / Adjustment Phase / Adaptation Phase Increase in cell size NOT in number Increase of enzyme and metabolic activity of bacteria LOG Phase / Exponential Phase Increase in growth rate (cell division/binary fission) Susceptible to antimicrobial agents (Best time to do AST) STATIONARY Phase / Plateau Phase No net growth rate (death = live cells) Increase death rate Cell death starts due to a. Toxin and waste products b. Depletion of nutrients c. Adverse environmental conditions DEATH Phase / Period of Decline No net growth rate (death = live cells)
STAINING PROCEDURE – stains the bacteria A. Direct – stains 1. Simple = 1 dye, Basic dye is used to stain bacteria Crystal Violet Methyl Red 2. Differential = 2 dyes (used to differentiate G + and - ) Gram Stain (both direct and Indirect – you can gram stain not only the specimen but also the colony in t he agar media) AFB 3. Special = Bacterial structure, Metachromatic granules B. Indirect/Relief/Negative – background – background is stained, for capsules a. India Ink / Borris Method b. Nigrossin Methods
Do NOT Gram Stain: 1. Chlamydia/Rickettsia – Chlamydia/Rickettsia – intracellular intracellular (stain cannot reach inside the cell) 2. Mycoplasma/Ureaplasma Mycoplasma/Ureaplasma – – cell cell wall less (walang kakapitan ang Cry stal Violet) 3. Spirochete – Spirochete – sin since ce it is very small, it can’t be gram stained. NOTE:
– a fluorescent dye that binds to bacterial DNA, which can be Acridine Orange – a used to stain Mycoplasma (no cell wall but with nucleic acid), since it has DNA. For Nucleic Acid. More sensitive than Gram stain. Fluorescent stain is more sensitive than gram stain and AFB. Any organism that cannot be gram stained, once gram stained, they are
– Gram stain for FUNGI Hucker’s Hucker’s Stain (Crystal Violet + Ammonium Oxalate) – (Gram +)
I. GRAM STAIN (PRESUMPTIVE NOT CONFIRMATORY) Purpose Primary Mordant Decolorizer
Reagents V (Crystal Violet) I (Iodine) A (95% Acetone-Alcohol)
Gram Positive Purple Purple Purple
Gram Negative Purple Purple Colorless
Counterstain
S (Safranin)
Purple
Red
NOTES: Mordant is alkaline in pH, increases affinity of the dye t o the organism. Decolorizer is the most critical step in gram staining (commonly mistaken) No MORDANT (Iodine): Gram (+) bacteria can be mistaken as Gram (-) No DECOLORIZER (Alcohol): Gram (-) bacteria can be mistaken as Gram (+) Gram (+) becomes Gram (-) 1. Over decolorization, Old dying 2. Use of acidic iodine as mordant 3. Penicillin, Omit iodine Gram (+) becomes Gram (-) 1. Under decolorization 2. Thick smear Gram Stain – Stain – General General Rule 1. All cocci are gram +ve, EXCEPT: / Gram Negative Cocci (NVM) Neisseria, Veilonella, Moraxella 2. All bacilli are gram -ve, EXCEPT: / Gram Positive Bacilli Mycobacteria, Corynebacteria Clostridia, Nocardia, Actinomyces Bacillus, Lactobacillus, Listeria, Erysiphilothrix 3. All spiral organisms are reported as Gram (-) 4. Yeasts and Fungi are Gram (+). Yeasts are differentiated to cocci based on SIZE. Size: Yeast > Cocci II. ACID FAST STAINING METHODS – METHODS – SCREENING not DIAGNOSTIC “Acid Fast” = Acid alcohol resistant not decolorized by acid alcohol retaining the RED color of CARBOLFUCHSIN due to the presence of MYCOLIC ACID (a long chain of fatty acid that makes Mycobacteria the bacteria with the highest amount of lipid) Mycobacteria = “Mataba “ Mataba at Mabagal” M abagal”
A. MYCOBACTERIA - Acid Fast Methods used for differentiation of Mycobacteria: Pappenheims – M. smegmatis vs. M. tuberculosis Baumgarten’s – M. leprae vs. M. tuberculosis Fite Faraco’s – M. leprae (hematoxylin) (Dx: Skin Biopsy) M. smegmatis – for uncircumcised patients M. leprae – causative agent of leprosy (Hansen’s disease) Specimen: Sputum, Urine, Stool (due to Intestinal MTB)
III. SPECIAL STAIN (such as Rickettsia, Chlamydia, Mycoplasma, Ureaplasma) a. Capsule – Negative stain b. Spore – Dorner, Wirtz Conklin, Schaeffer Fulton c. Metachromatic Granules – Albert’s, LAMB, Neisseria d. Flagella – Leifson, Gray’s e. Nucleic Acid – Fuelgen f. Polar bodies – Wayson g. Rickettsia – Gimenez, Macchiavelo h. Spirochetes – Levaditi, Fontana, Tribondeau Note: (Non Staining Method) STRING TEST = uses 3% KOH. Presence of STRING LINE = Gram (-) Bacteria.
B. NOCARDIA and CRYPTOSPORIDIUM – Modified Acid Fast = uses 1% H2SO4 as Decolorizer instead of Acid Alcohol = Cold method (no heat required)
TYPES OF MICROSCOPY Nocardia Specimen: Sputum (since Nocardia is an agent of Pneumonia) Cryptosporidium Specimen: Stool (mostly for patients with HIV)
ACID FAST: Purpose
Primary (10 min) Start timing when Steam appears Mordant (3 min) Decolorizer Counterstain (30 sec) Result
Ziehl-Neelsen (Hot) (C-A-M) Carbolfuchsin
Kinyoun (Cold) (C-A-M) Carbolfuchsin
Heat
Phenol, Tergitol
3% Acid Alcohol Methylene Blue
3% Acid Alcohol Malachite Green
AFO – Red NAFO – Blue
AFO – Red NAFO – Green
RhodamineAuramine (Fluorochrome) AuramineRhodamine
0.5% Acid Alcohol 0.5% KMNO4 Quenching Agent AFO (+) - Yellow Fluorescence NAFO – No fluor,
NOTE:
Ziehl-Neelsen: Best Method Kinyoun : Used in tissue samples Rhodamine Auramine: Most sensitive method Heating removes the fat allowing the penetration of the stain to the cell wall Acid Alcohol Composition: (HCl + 95% Ethyl Alcohol); For Nocardia: 1% H2SO4 KMNO4 (Quenching Agent) – absorbs fluorescence LED Fluorescent Microscopy – new fluorescent stain, more sensitive than Auramine Rhodamine
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Brightfield Darkfield – motility of Spirochetes; confirm Primary Syphilis Phase Contrast – used for living cells and inclusion body ( virus and Chlamydia can produce inclusion body); also used for HLA TYPING Fluorescent For Bacteria: Acridine Orange: Red; Aura-Rauda: Yellow (PEPTIDOGLYCAN) For Fungi: Calcofluor White binding in the CHITIN CELL WALL For Serology: Immunofluorescent Test Electron – with the highest magnification a. TEM – Transmission (internal structure) requires a stain: Phosphotungstic Acid (Negative Stain) b. SEM – Scanning (external/surface structure)
TYPES OF CULTURE 1.
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Pure Culture – most important! Where Identification and AST is done. a. Streak Plate (best method) b. Pour Plate c. Selective Medium d. Animal Inoculation Mixed Culture – 2 or more bacterial species Stock Culture – for Quality control; stored at - 20’C/Freezer Working Culture – 4’C, from Stock Culture
According to Consistency: a. Liquid (broth) – used to increase number o f bacteria, mostly for swab specimens since swab sp. have only small amount of bacteria b. Semi-solid = 0.5 – 1% agar (motility), for SIM. (Motile = Hazy; NonMotile = Clear) c. Solid = 2-3% agar (plated media) d. Biphasic = Both liquid and solid ( Castaneda) = Blood Culture media for Brucella
Types of Culture Media: 1. General Purpose Media – NON FASTIDIOUS a. BAP (Blood Agar Plate) good for hemolysis study Contains X Factor (HEMIN – Heat stable) for both: Gram (+) White Dry Colony Gram (-) Gray Moist Colony Sheep’s Blood – Streptococcus Horse Blood – Haemophilus hemolyticus/parahemolyticus Human Blood – beta hemolysis of Gardnerella vaginalis b. NA (Nutrient Agar) 2. Enriched Media – FASTIDIOUS a. CAP (Chocolate Agar Plate) for culture only not for hemolysis study Contains X and V Factor (NAD – Heat labile) Does not contain Chocolate but LYSED RBC Horse Blood – best source of blood for CAP good for Neisseria b. BCYE 3. Enrichment (Broth) – enhance the growth of bacteria Increase the LAG phase of NORMAL FLORA Decrease the LOG phase of PATHOGEN a. Selenite F, APW, THIO 4. Differential a. BAP – differentiates alpha, beta, gamma hemolysis b. Mac – differentiates lactose from non-lactose fermenters (Important for differentiation of pathogenicity of Enterobacteriaciae, NLF are pathogenic than LF) c. EMB, XLD, HEA 5. Selective (inhibitory agents) pathogenic organisms are needed in a non-sterile specimen a. TCBS – Vibrio (Stool) b. TMA (Thayer Martin Agar) – Neisseria c. CBAP - Campylobacter Inhibitory Agents – ANTIBIOTICS DYES, BILE SALT = Inhibits Gram + (For Gram Neg only) a. Mac – contains crystal violet and bile salt (selective to gram -) b. EMB – contains dyes (Eosin and Methylene Blue) ALCOHOL (PEA) = Inhibits Gram – (For Gram Pos only) a. CNA (Collistin Nalidixic Acid) Common Culture Media: 1. PEA 2. COLUMBIA CNA
Gram (+) bacteria Gram (+) bacteria
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BASITRACIN CAP CYSTINE BLOOD GLUCOSE AGAR CYSTINE TELLURITE BLOOD AGAR CYSTINE TRYPTICASE AGAR CHARCOAL CEPHALEXIN BLOOD AGAR BCYE McCOY TSB THIOGLYCOLLATE Potato Blood Glycerol Agar
H. influenzae Francisella C. diptheriae Neisseria (Confirm) B. pertusis Legionella pneumophila C. trachomatis Brucella spp (Aerobes) Aerobes/Anaerobes B. pertusis
NOTE:
TSB is mostly for Aerobes. Brucella spp. are Obligate Aerobe. Brucella causes Brucellosis, Endocarditis; Specimen: Blood; Media: Castaneda) Thioglycollate is for both Aerobes and Anaerobes Glycerol in PBGA is made up of egg = LJ Medium
SPECIMEN HANDLING AND COLLECTION Types of Specimen Sterile None Sterile Aerobic (24 hours); Anaearobic (48 hours) Collection Swab Cotton – toxic for NEISSERIA, good for VIRUS (Countertoxicity: Charcoal) Calcium alginate - toxic for VIRUS, good for NEISSERIA Bronchial washing – for AEROBIC culture Needle aspiration – for both AEROBIC and ANAEROBIC Catheterization – for sterile urine Intubation – for gastric samples ( H. pylori = Urea Breath Test) Delays Refrigerator except: 1. CSF – immediately processed a. Room Temp – transport temperature b. 35’C – storage temperature (incubator) 2. Blood 3. Urogenital Swab of N. gonorrhea – sensitive to cold temperature (do not ref) 4. Boric acid – preservative for URINE culture 5. Cary Blair – rectal swab
Transport Medium: 1. Cary Blair – stool pathogens (for enteric pathogens, VIBRIO) 2. Stuart’s –Viral Transport Medium 3. Amies – respiratory 4. Transgrow – Neisseria 5. JEMBEC – Neisseria 6. Todd Hewitt – GROUP B Strep S. agalactiae (vaginal swab)
CLINICAL SPECIMEN 1.
Biologic Safety Cabinet HEPA Filter – air sterilization, holds bacteria in the air Negative Pressure – takes infectious air outside the BSC Note: Not required in AFS, but for culture and sensitivity 1. Class I air velocity 75 linear feet/min with product (culture) contamination exhaust air through ONE HEPA filter 2. Class II (Vertical Laminar Flow) air velocity 75-100 linear feet/min no product (culture) contamination exhaust and recirculated air through TWO HEPA filters MUST for MICRO lab/hospitals (tertiary) a. IIa = exhausts air inside the room b. IIb = exhausts air outside the building 3. Class III Maximum protection Supply and exhaust air through TWO HEPA filters For BSL Level IV (viruses)
(+)BAP (+)CAP (+)MAC Gram (-)
No risk
Biosafety Level II
Moderate risk
Biosafety Level III
High risk (With Treatment)
Biosafety Level IV
High Risk (No Treatment)
M. gordonae, B. subtilis Y. pestis B. anthracis Mycobacteria Brucella Francisella, Molds Viruses
Note: Brucella is a FASTIDIOUS organism therefore hard to culture, requiring 21 days.
BSC Class II
Note: B. anthracis and Y. pestis are agent of bioterrorism yet easily destroyed by penicillin. Francisella and Brucella are laboratory acquired infections
(-)BAP (+)CAP (-)MAC Neisseria gonorrhea Haemophilus influenzae
After 7 days: Negative th Blood Culture Contaminant: Staph. epidermidis (5 day (+)); 7 Days before reporting negative: Bacteremia (typhoid) 21 Days before reporting negative: Brucellosis, Endocarditis, SBE (HACEK)
BSC Class I
BSC Class III
(+)BAP (+)CAP (-)MAC Gram (+)
Neisseria g. = Genital specimen Haemophilus i . = respiratory and CSF specimen
Classification of Biologic Agents (Risk Level of Organisms): Biosafety Level I
Blood (BHIB) requires TWO to THREE blood culture to rule out bacteremia 1:10 (1ml of Blood to 10ml of Broth media) Antibiotic Removal Device (ARD) – this will remove the antibiotic the patient is taking Collection Time: Before antibiotic treatment During acute stage of infection SPS – anticoagulant (0.25% SPS); needed since the clotting of blood will trap the bacteria Anti-complimentary and anti-phagocytic: preventing hemolysis Neutralizes: aminoglycosides (antibiotics) and bactericidal effect of serum Inhibits: G. vaginalis, Neisseria, S. monoliformis, P. anaerobius NOTE: 1% GELATIN counteracts SPS Bacterial Growth in Blood: (+) Hemolysis, turbidity, pellicle, bubble formation If (+), Subculture in: BAP, CAP, Mac
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Urine Catheterized (bedridden), midstream (female), suprapubic (anaerobic culture) Quantitative Technique / Colony Count (BAP for (G+), Mac for (G-)): only applicable for MIDSTREAM Collection >100,000 CFU – significant for UTI <10, 000 CFU – not significant
Agents causing UTI: (+)BAP (+)Mac = Gram NEG= (#1) E. coli, (#2) Proteus (+)BAP (-)Mac = Gram POS= S. saprophyticus, Enterococcoci, C. urealyticum
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CSF – not refrigerated; immediately processed Neisseria meningitidis and Haemophilus influenzae Culture Media: CAP (most important culture media for CSF) BHI(to enhance the growth of the organism since they are FASTIDIOUS) BAP, Mac India Ink method: CAPSULE of Cryptococcus meningitis Latex: CAPSULAR ANTIGEN of Cryptococcus meningitis
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Vaginal, Urethral Swab CAP Modified Thayer Martin Gram Stain
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TB culture Requires BSC Level II NALC-NaOH (gold standard) NALC – digestant for sputum (N-acetyl L-cysteine) NaOH (2-4% NaOH) – digestant and decontaminant Oxalic Acid – used when specimen is contaminated by Pseudomonas, such as URINE and STOOL Clorox (Anti-formin) – cannot clear Mycobacteria, but can destroy virus. CENTRIFUGE: for 15 mins at 3000rpm ( 4’C Temp. of centri for TB culture = “Ref Centrifuge” to prevent aerosol) Culture Media: Lowenstein-Jensen (Green) – best culture medium (test tube) for TB Middlebrook 7H11, 7H10 - can be used as AST media TAT before reporting as NEGATIVE: LJ Medium = 8wks (through incubation at 37’C) BACTEC: 2-3 days DNA Test: 2-3 hours (+) 2-3 weeks, growth is seen NOTE: BACTEC = uses radiometric method
NOTE: Centrifuge Urine and CSF for 2000rpm for 10 minutes. Sediments are used for culture.
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Wound Gram (+): S. aureus (#1 cause) Gram (-): Pseudomonas, Vibrio, Enterobacteriaciae Culture Media: Thioglycollate (Anaerobes causing wound infection, FOUL odor) Gram stain BAP, Mac
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Stool – NOT Gram Stain - Selective Media is needed for Pathogenic Organisms: Aeromonas, Campylobacter, Salmonella, Shigella, Vibrio MacConkey, BAP + ampicillin CBAP, SSA, Selenite F TCBS, Alkaline Peptone, HEA Tests: Oxidase Test Biochemical Test (Screening) Serologic Typing(Confirmatory): E.coli, Salmonella, Shigella, Vibrio
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Respiratory – (sputum, NPS) BAP (S. pneumoniae = Rusty sputum) BCA (Basitracin Chocolate Agar): H. influenzae Mac, GBA (Gentamicin BA) Amies, Do gram stain and AFS
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Throat Swab – Sore throat BAP (Strep for hemolysis) Modified Thayer Martin (Neisseria gonorrhea, but still best in genital)
STERILIZATION AND DISINFECTION
Sterilization – standard for microbiology; both the pathogenic and nonpathogenic organisms are destroyes Disinfection – only the pathogenic is destroyed Moist Heat – destroy bacteria through coagulation of protein Quality Control: Moist Heat: Bacillus stearothermophilus o Dry Heat: Bacillus subtilis o
STERILIZATION METHOD – MOIST HEAT 1. AUTOCLAVE – steam under pressure, BEST sterilization procedure Autoclave tape – indicator Bacillus stearothermophilus – used for quality control of autoclave (WEEKLY BASIS) 121’c at 15lbs/psi for 15 min Culture media, bandages, gauze 2. INSPISSATION 75-80’C for 2 hours on 3 days
3. 4. 5.
Note: 1. 2.
TYNDALLIZATION 100’C for 30min on 3 days BOILING – non sporicidal, only kills vegetative. 100’C for 30min PASTEURIZATION – only the pathogens are destroyed in t he milk sample 63'C for 30min / 72'C for 15secs 75,000 –15,000 – count of bacteria before and after milk pasteurization 10,000 - count of bacteria for certified milk Pseudomonas syncyanea – causes BLUE MILK Flavobacterium synxanthium – causes YELLOW MILK Tests: Phosphatase Test – Test for pasteurization Result: (-): Success of Pasteurization Methylene Blue Reduction Test – detects the presence of bacteria in milk sample Result: (+) Colorless; (-) Blue Litmus Milk Test – detects the pH of milk
Autoclave, Inspissation and Tyndallization are SPORICIDAL Inspissation and Tyndallization are forms of Fractional Crystallization requiring 3 days. Day 1 – Destroys Vegetative Cells Day 2 – Destroys Spores Day 3 – All cells are destroyed
6. 7.
DISINFECTANT – ANTISEPTIC 1. 10% Sodium Hypochlorite (Clorox) – best disinfectant, destroys HIV and HBV 2. Iodophor – combination Iodine and Detergent, best skin antiseptic (sporicidal) 3. 70% Ethyl Alcohol – non sporicidal 4. 1% Silver Nitrate – (eyedrop) prevents Prophylaxis; prevents gonococcal opthalmia neonatorum 5. 3% Hydrogen Peroxide – used in catalase test; antiseptic 6. Dyes – inhibits gram positive 7. Formaldehyde – sporicidal, preservative for tissue samples 8. Glutaraldehye – sporicidal, sterilize surgical instruments 9. 5% Phenol (Carbolic Acid) – standard (“benchmark”) disinfectant 10. Lysol (Cresol) – disinfectant 11. Zephiran (Benzalkonium chloride) – Merthiolate; antiseptic 12. Quats – (Quarternary ammonium compound) inactivated by organic materials
ANTIMICROBIAL AGENTS 1. 2. 3.
STERILIZATION METHOD – DRY HEAT 1. Hot Air Oven = Drying Dry heat method Bacillus subtilis – used for quality control of Oven Dry hot air at 170-180’C for 2 hours Glasswares, cotton swabs, metallic instruments, oils, powders 2. Incineration = Waste Disposal 3. Cremation = Control communicable disease 4. Flaming = Needles, burning organism into ashes 5. Gas-Ethylene Oxide = Heat Labile Others: 1. Cold Temperature/Freezing – preserve reagents, bacteriostatic – inhibits the growth of bacteria 2. Lyophilization/Freeze Drying – BEST to preserve microbial cultures 3. Osmotic Pressure – foods (bacteriostatic) 4. Dessication – foods 5. Ultraviolet Light – acts on the DNA of the organism (air and water) and leads to
Ionizing Radiation – disposables (gloves, microwave oven, catheter) Filtration (air and water) a. HEPA filter – filter for Air (0.3 um) b. Cellulose Membrane – Liquid (0.22um)
4. 5.
A.
Antagonistic = 1>2 (The effect of 1 drug is better than the combined effect of 2 drugs) Synergistic = 2>1 (The combined effect of 2 drugs is better than the effect of 1 drug) MIC = Minimum Inhibitory Concentration – Lowest concentration of drug to kill bacteria MBC/MLC = Minimum Bactericial/Lethal Beta Lactamase – if the bacteria is found to be resistant to Penicillin, perform Beta Lactamase test)
Cell Wall Inhibitors Broad Spectrum (Inhibits both Gram + and -) 1. Penicillin (Penicillum notatum) – inhibits Peptidoglycan synthesis 2. Cephalosporin (Cephalosporium) 3. Cycloserine 4. Imepinem, Carbapenems 5. Penicillinase Resistant Antibiotics = Methicillin, Cloxacillin, Nafcillin Narrow Spectrum 1. Vancomycin (Streptomyces) = inhibits Gram (+) ONLY; MRSA = Penicillin (R); Vancomycin (S) 2. Basitracin (Bacillus subtilis); inhibits Gram (+) ONLY
B.
Cell Membrane Inhibitors 1. Colistin – Inhibits GRAM (-) ONLY 2. Polymyxin (Bacillus subtilis) - Inhibits GRAM (-) ONLY 3. Amphoteracin B (Streptomyces) – Anti-FUNGAL 4. Nystatin – Anti-FUNGAL NOTE: Antifungal Agents – targets the CELL MEMBRANE
C.
Ribosomes (Protein) Inihibitors (All Broad Spectrum) 1. Aminoglycosides (-cin, Gentamicin) – antibiotic that has the HIGHEST DRUG RESISTANCE, affected with the addition of Calcium and Magnesium in Mueller Hinton Agar 2. Tetracycline 3. Cloramphenicol 4. Erythromycin (Macrolide) – “wonder drug” 5. Clindamycin – Antibiotic associated enterocolitis affecting Clostridum difficile NOTE: Pseudomonas aeruginosa – bacteria that has the highest drug resistance, also #1 seen in ICU (nosocomial)
D.
Nucleic Acid (DNA) Inhibitor 1. Mitomycin, Quinolones (-floxacins) – acts on the DNA 2. Metronidazole – Anti-PROTOZOA, Anti ANAEROBES 3. Sulfonamide-Trimetophrim (SXT) - inhibits FOLIC ACID (needed for DNA Synthesis) 4. Rifampin – anti TB Drug
I. Methods of Antibiotic Susceptibility Testing 1. Micro/Macrobroth Dilution recommended for ANAEROBIC BACTERIA reference method for MIC and MBC (Antibiotic is being diluted) as dilution increases, the concentration of the antibiotic decreases 2. Agar dilution = many organisms vs single drug 3. Disk Diffusion = one organism vs multiple drugs (MOST COMMON) 4. E Test (Epsilometer Test) antibiotic strip diffusion MIC test uses filter paper/strip incorporated with DECREASING concentration of antibiotic 5. VITEK/Automated System both identification and susceptibility test gives you the exact amount of antibiotic to inhibit the growth of organism Errors: No Identification result : Do the manual/conventional method Doubtful/Unfamiliar of the ID result: Endorse/refer to the supervisor Misidentification of result: Do confirmatory test using other method 6. Microstat Walk-Away System = combination of Identification, Susceptibility
7.
Disc Elution Test = Susceptibilty test for Mycobacteria Antibiotic is FIRST applied in the agar, B acteria is the LAST to be a pplied (unlike in Kirby Bauer vice versa). Requires 1 drop of inoculum in t he four quadrants (S) = No Colony (R)= With Colony MDR-TB (Multi-Drug Resistant TB) = Mycobacteria that is resistant to o primary drugs ISONIAZID and RIFAMPICIN XDR-TB (Extensively Drug Resistant TB) = Mycobacteria that is resistant o to ALL DRUGS + QUINOLONES
II. Antibiotic Susceptibility Testing (AST) Media - All are CLEAR Media, making zone o f inhibition and colonies easily seen - BAP cannot be used since it’s a dark medium a. MHA – general AST media b. MHA + 2% NACL – MRSA c. MHA + 5% Sheep’s Blood – Strep d. Heamophilus Test Medium (MHA + Yeast Extract) e. GC Agar (Gonococci Agar) – Neisseria f. Middlebrook 7H10 – Mycobacteria III. Disk Diffusion – Kirby Bauer (Semi Quantitative) STANDARD INOCULUM 1.5 x 108 MEDIUM Mueller Hinton Agar (MHA) pH 7.2 – 7.4 DEPTH 4mm (standard thickness of agar) CONDITION Aerobic, No CO 2 (to prevent increase in pH) TEMPERATURE 35-37’C (MRSA-35’C) INC. TIME 16-18 hours STANDARD 0.5 McFarland (1% H 2SO4 and 1.175& BaCl 2) ANTIBIOTIC DISK 6mm NOTE: Petroff-Hauser = Bacterial Counting Chamber McFarland for Fungi: 2.0 IV. Zone of Inhibition 6mm = Resistant Standard distance between 2 antibiotic disk = >15mm (to avoid overlapping of zone of inhibition) <15 min = antibiotic disks should be applied on the agar after streaking. Delay causes SMALLER ZONE Antibiotic Disk = responsible for the zone of inhibition NOT the organism Presence of Swarming = Ignore. Continue measuring the zone of inhibition. Presence of Double Zone = OUTER ZONE is measured not the inner zone. Presence of Colony Inside the Zone do gram stain
False Resistant 1. Heavy inoculum (The higher the bacteria, the smaller the zone of inhibition) 2. Thick Medium 3. Delay in Disc Application (should be applied <15min) 4. Ca/Mg – aminoglycoside (P.aeruginosa) 5. Thymine-Thymidine-SXT (Enterococci) False Sensitive 1. Light inoculum (The lesser the bacteria, the larger the zone o f inhibition) 2. Thin medium 3. Delay in Incubation (growth of organisms are affected 4. Presence of CO2 (Increased pH) – False sensitive in Tetracycline
GRAM POSITIVE COCCI I. STAPHYLOCOCCI CATALASE (+) Staphylococci I COAGULASE
(+) S. aureus
Perform AST for the following Organisms: 1. Pathogenic Organism 2. Drug Resistant Organism 3. Opportunistic Organism QUALITY CONTROL Quality Control – routine (internal QC) - checking media and reagents with specific organisms Quality Assurance – external QC, annually QC Frequency: a. Daily QC Oxidase, Catalase, Gram Stain Incubator, Ref/Freezer, Water Bath b. Each use Gas Pak jar, ONPG c. Weekly QC Antibiotic, Autoclave, Biochem d. Monthly (30-Day) QC New drugs and reagents e. ATCC Reference strains for QC f. -20’C or -70’C Stock culture storage -70’C (Virus and PCR) g. 2-8’C Working cultures storage
(-) Streptococci
(-) MOD. OXIDASE/BACITRACIN
+/S Micrococcus
-/R Coag. Negative Staph I NOVOBIOCIN
S S. epiderdimis
R S. saprophyticus
A. Diagnostic Tests: 1. CATALASE TEST (Presumptive Test) Catalase enzyme on 3% H 2O2 Performed on slide. Not performed on BAP since blood contains catalase = false positive (+) Gas bubbles (+) Staphylococcus; (-) Streptococcus 2.
COAGULASE TEST (Confirmatory Test for S. aureus) Bacterial colonies Clot ( 4 Hours) a. Slide (Screening) – BOUND coagulase b. Test Tube (Confirmatory) – FREE coagulase Medium: Rabbit’s Plasma with EDTA (not citrate because there are some bacteria that can utilize citrate, e.g. Enterococci, Campylobacter ) (+) Staphylococcus aureus
3.
MANNITOL FERMENTATION TEST Mannitol Salt Agar (7.5% NaCl) Indicator: Phenol Red (+)Yellow (S. aureus) - Acid Production (-) Red
4.
DNASE TEST Two Methods: 1. Toluidine Blue – pink zone (Presence of DNAse) Methyl Green – clear zone (Presence of DNAse) 2. HCl Precipitation – no precipitation after 1N HCl (Presence of DNAse) DNAse (+) = Clear Zone DNAse (-) = No Clearing Bacteria used as a POSITIVE CONTROL for DNAse Test: 1. Staphylococcus aureus (Gram + cocci) 2. Serratia marcescens (Gram – bacilli)
5.
NOVOBIOCIN TEST (5 units) CNS differential test ID. of S. saprophyticus R = <16mm
6.
MODIFIED OXIDASE TEST Reagent = Tetramethyl p-phenylene diamine dihydrochloride in DMSO (Dimethyl Sulfoxide) (+) Blue/Purple ( Micrococcus luteus ) (-) No color change
7.
STAPH A COAGGLUTINATION TEST A means Protein A that is found at the cell wall of S. aureus. Staph aureus (cowan strain) with protein A as inert particles to which antibody (Fc fragment) binds Detects specific bacterial Ag (Strep. Pneumoniae, N. meningitides, N. gonorrhea, H. influenzae) Micrococcaceae Micrococcus Staphylococcus Oxidative Fermentative O/F Test + Modified Oxidase S R Basitracin R S Furazolidone R S Lysostaphin Note: Stomatococcus: Mod. Oxidase (-), Lysostaphin (R) and Furazolidone (R)
B. Staphylococcus aureus Properties: Protein A – cell wall, antiphagocytic, virulence Enterotoxin – food poisoning Beta Hemolysin Leukocidin – Panton Valentine Exfoliatin (epidermolysis) – SSS TSST-1 – Toxic Shock Syndrome Beta Lactamase – drug resistance DNAse – dissolves clot Hyaluronidase – spreading factor Gelatinase Lipase – fat splitting enzyme C. Staphylococcus aureus Identification: Yellow orange colony – lipochrome Catalase (+), Coagulase (+), Oxidase (-), Nitrate and VP (+), Gelatin (+), PYR (-) Staph like Organisms S. intermidius S. lugdunensis S. haemolyticus
SIMILARITY TO S. aureus Slightly Coagulase (+) Slightly Coagulase (+) Beta Hemolytic
DIFFERENCE VP (-) (Acetoin) PYR (+) Coagulase (-)
NOTE: It’s is better to perform TEST TUBE METHOD than slide method because S. intermidius and S. lugdunensis are Coagulase (-) in t est tube method for coagulase test. Diseases : #1 SKIN INFECTIONS: Carbuncles, furuncles, folliculitis, cellulitis, impetigo, skin scalded syndrome (SSS), TSS (Tampons) #1 WOUND, #1 OSTEOMYELITIS, #1 NOSOCOMIAL Bacteremia, endocarditis, Food posining Lab Diagnosis: 1. Gram Stain – can be applied directly in the wound swab 2. Culture BAP (Slightly beta haemolytic, pinhead colony) Vogel-Johnson (brown to black colony because s. aureus can reduce Tellurite present in VJ agar; Corynebacterium can also reduce Tellurite. Difference: S. aureus is cocci while Corynebacterium is bacilli) Chapman, Tellurite Glycine, P Agar, PEA, Columbia CNA 3. Catalase (+) Coagulase (+) 4. Mannitol Fermentation Test (+): Yellow 5. DNA Hydrolysis Test 6. Latex Agglutination test for protein A (Co nfirmatory Test) : (+)Agglutination
D. Coagulase Negative Staph: 1. STAPH. EPIDERMIDIS Skin flora, Blood culture contaminant Causes PROSTHETIC HEART VALVE, ENDOCARDITIS Novobiocin Sensitive, Non Hemolytic, O xidase (-) 2.
STAPH. SAPROPHYTICUS Causes UTI, sexually transmitted Novobiocin Resistant
II. STREPTOCOCCI ALPHA HEMOLYSIS I OPTOCHIN (S) S. pneumoniae
Note: S. aureus and S. epidermidis are both Novobiocin sensitive. S. saprophyticus is the only Novobiocin resistant S. aureus Colony Catalase Coagulase Mannitol Novobiocin DNAse Phosphatase Gelatinase
Yellow + + + S + + +
Coagulase Negative S. epidermidis S. saprophyticus White White + + +/S R + + +
(R) Group D Strep S. viridans I BILE ESCULIN (+) Group D Strep
(-) S. viridans
BETA HEMOLYSIS I BACITRACIN (S) Group A Strep
(R) Group B, C, D, F, G Strep I BILE ESCULIN (+) Group D Strep
(-) Group B (CAMP +) Group C, F, G (SXT +)
GAMMA HEMOLYSIS I BILE ESCULIN (+) Group D Strep I 6.5% NaCl PYR (+) Enterococci
(-) Non-Enterococci
A. Diagnostic Tests: (Presumptive Test Only) 1. BACITRACIN SUSCEPTIBILITY / TAXO A (0.04 Units) ID of S. pyogenes (+) Zone of Inhibition 2.
PYR (L-pyrrolidonyl B-napthlamide) ID of S. pyogenes, Enterococcus Rgt: p-dimethylaminocinnamaldehyde (+) Red
3.
CAMP TEST CAMP factor of S. agalactiae synergistic reaction to beta lysine of S. aureus (+) Arrow head zone beta hemolysis
4.
HIPPURATE HYDROLYSIS TEST ID of S. agalactiae Rgt: Sodium Hippurate and Ninhydrin (+) Purple S. agalactiae Listeria Campylobacter (-) Colorless, Pink
5.
OPTOCHIN TEST / TAXO P (5 units) ID of S. pneumoniae 5ug ethylhycrocupreine HCl (Taxo P) (+) >14 mm zone of inhibition (S. pneumo) (-) <13mm zone or no zone (S. mitis)
6.
BILE SOLUBILITY TEST Incubate: 35’C for 30 mins Rgt: Sodium Desoxycholate (Bile salt) – able to destroy gram positive bacteria BAP (10% Bile Salt) (+) Lyzed Colony (S. pneumoniae) (-) Intact Colony (S.viridans / E. faecalis) Tube Method (2% Bile Salt) (+) Clear (-) Turbid
7.
BILE ESCULIN HOH TEST 40% Bile Ferric NH4 Citrate reacts with esculetin
8.
VANCOMYCIN RESISTANT ID of Pediococcus/Leuconostoc (Streptococcus-like organisms that are resistant to Vancomycin)
Tests for Differentiation LEUCINE AMINOPEPTIDASE (LAP) MRS BROTH 9.
Pediococcus (+) Red (-) No Gas Production
Leuconostoc (-) Yellow/No Color change (+) Gas in Durham Tube
PYRUVATE BROTH Incubate: 35’C for 48 hours (+) Yellow (E. faecalis) (-) Green (E. faecium)
NOTE: All of the above are only PRESUMPTIVE test. The CONFIRMATORY test for Strep is LANCEFIELD TEST (Serologic Typing) except for S. pneumoniae since it doesn’t have lancefield classification. The CONFIRMATORY test for S. pneumoniae is NEUFELD QUELLUNG TEST.
B. Streptococcus Characteristics: Gram (+) cocci in chain, pairs Catalase (-), “Pinpoint” colonies Oxidase (-) (Remember that Oxidase (+) are usually for Gram Negative) Facultative Anaerobes Capnophilic (5-10% CO2) Medium of Choice: S heep’s Blood Agar Selective Medium: PEA C. Streptococcus Classifications: 1. Smith and Brown’s Classification a. Alpha Streptococcus Incomplete Hemolysis (+) Greenish Zone S. pneumoniae, S. viridans Note: ALPHA PRIME – a small zone of alpha hemolysis surrounded by zone of beta hemolysis after refrigeration b.
Beta Streptococcus (Common) Complete Hemolysis (+) Clear/Colorless Zone S. pyogenes, S. agalactiae, Grps. C F G
c.
Gamma Streptococcus
2.
Lancefield Classification (CONFIRMATORY) - Carbohydrate on cell wall (Grp. A, B, C …) a. GROUP A Strep (Streptococcus pyogenes) pus forming, flesh eating bacteria requires anaerobic incubation to detect hemolysis Characteristics: M protein – found at the cell wall, antiphagocytic, virulence factor Streptolysin O – O2 labile, Ag, Sub surface (needs Anaerobic incubation to detect hemolysis) Streptolysin S – O2 stable, Non Ag Erythrogenic toxin – causing scarlet fever Streptokinase – dissolves clot (treatment to prevent AMI) Hyaluronisade – spreading factor Bacitracin Sensitive Diseases: 1. Pharyngitis – “Sore throat” (Specimen: Throat swab) 2. AGN, RF (Rheumatic Heart Disease) - since it cross reacts with myocardial antigens 3. Scarlet Fever a. Dick’s Test (red) – skin test where toxin is given b. Schultz-Charlton (rash fade) – immunity test 4. Erysipelas 5. Impetigo 6. Wound, Burn 7. Toxic Shock Syndrome, Pyoderma, Necrotizing Fascitis Lab Diagnosis: 1. Gram Stain – Gram (+) cocci in chain 2. Culture – BAP (BH) 3. Catalase – Catalase (-) (no gas bubbles) 4. Basitracin (Taxo A) – sensitive (any zone) 5. SXT Test – resistant 6. PYR Test – PYR (+) Red 7. Lancefield Typing – Group A
NOTE:
A Protein = S. aureus M Protein = S. pyogenes Dick’s Test = S. pyogenes Schick’s Test = Corynebacterium diptheriae Erysipelas/Scarlet Fever= S. pyogenes Erysiperloid = Erysipelothrix
b.
1. 2.
GROUP B Strep (Streptococcus agalactiae) Beta Hemolytic Vaginal flora, URT #1 Neonatal Meningitis (acquired through vaginal delivery), Septicemia Lab Diagnosis: CAMP Test: (+) Arrow Head zone of BH Hippurate Hydrolysis Test: (+) Purple c.
GROUP C, F, G Strep Animal pathogens that cause endocarditis ( Specimen: Blood ) Beta Hemolytic, Basitracin Resistant SXT Sensitive Group C: S. equimilis, S. equi, S. dysagalactiae Basitracin S R R
1.
2.
SXT R R S
Organism Group A Strep Group B Strep Group C, F, G Strep
d. GROUP D Strep Enterococcus E. faecalis, E. faecium, E. durans Drug resistant (VRE), UTI Non-Enterococcus S. bovis – colon cancers; S. equinus Easier to treat than Enterococcus (Penicillin Sensitive) Lab Diagnosis: Bile Esculin Hydrolysis Test (+) Blackening; Bile Esculin Medium Differentiates Group D from other Strep Presumptive test for Group D
D. Streptococcus pneumoniae: Gram (+) diplococci, “ Lancet Shaped” No Lancefield classification Alpha haemolytic, Virulence: Capsule Nasopharynx and Oropharynx Inhibited by OPTOCHIN; Bile Soluble
F. Pediococcus/Leuconostoc (Strep-like Organisms) Vancomycin resistant test – differentiate Pediococcus from Strep viridans Note: Leuconostoc is S. viridans-like/ Aerococcus is Enterococcus-like
Diseases: 1. #1 Adult Bacterial Meningitis ( Specimen: CSF ) 2. Pneumococcal pneumonia = (“Rusty Sputum”) 3. #1 Otitis Media Lab Diagnosis: 1. Gram Stain – Gram (+) diplococci 2. Culture – Gentamicin Blood Agar (alpha, mucoid colony) 3. Optochin (Taxo P) - >14mm zone 4. Bile Solubility a. 10% Sodium Desoxycholate (BAP) (+) Lysis Colony b. 2% Sodium Desoxycholate (tube) (+) Clear 5. Mouse Virulence Test - death 6. Francis Test – skin 7. Neufeld Quellung Test – capsular swelling (confirmatory) NOTE:
#1 Neonatal Meningitis = S. agalactiae #1 Adult Meningitis = S. pneumoniae
Species: 1. S. mitis (mitior) 2. S. sanguis - subacute endocarditis (SBE) acquired through dental procedure 3. S. mutans - dental plaques/caries Enterococcus + + + R R + +
Beta Hemolytic Strep TESTS Basitracin SXT CAMP PYR Bile Solubility
Pediococcus R + + (+) Red
Group A S R + -
Leuconostoc R + + (+) Gas -
Group B R R + -
Aerococcus S +/+/+ N/A N/A
Group C, F, G R S -
G. Abiotrophia spp Nutritionally variant streptococci (NVS) Strep that will not grow in BAP or CAP Requires Vit. B6 (pyridoxine); Staph streat test (+)
GRAM NEGATIVE COCCI
E. Streptococcus viridans: Not classified under LANCEFIELD (same as S. pneumoniae) OPTOCHIN Resistant, Bile Insoluble Normal flora of URT, GIT, GUT
Bile Esculin 6.5% NaCl PYR Penicillin Basitracin Growth 45’C Growth 10’C
TESTS Vancomycin Bile Esculin PYR 6.5% NaCl MRS Broth LAP
Non Enterococcus + S R + -
Genera included: Aerobic: Neisseria and Moraxella Anaerobic: Veilonella Gram (-) Intra (extra) diplococci Oxidase (+) (Presumptive test for gram negative organism) , Catalase (+) Best Media: CAP (Requiring 5-10% CO2) Charcoal is added in culture media to remove the toxicity of cotton Pathogenic: N. gonorrhoeae, N. meningitides Pigmented Neisseria: N. subflava, flavescenes (Yellow color due to Flavin)
A. Neisseria gonorrhoeae Aka: “CLAP” (Since clapping requires 2 hands = diplococci) Kidney (coffee) bean shaped in PMN Oxidase positive (PRESUMPTIVE) and ferment glucose (dextrose) (CONFIRMATORY) Virulence – “pili” (N. meningitides – capsule and endotoxin) Diseases: gonorrhea, opthalmia neonatorum, Fitz-Hugh Curtis, PPNG (Penicillinase Producing Neisseria gonorrhea); salphingitis, epididymitis
Laboratory Diagnosis: 1. GRAM STAIN – the presence of any diplococci inside or outside a PMN is an indicative of Neisseria gonorrhea 2.
CULTURE: CAP (+); BAP (-) (NON STERILE) Selective Media Chococolate Agar Containing Media: 1. Thayer Martin Agar – VCN 2. Modified TMA – VNCT – Trimethoprim lactate 3. Martin Lewis – VCAT (Anisomycin)
Yeast Extract Agar (Clear Media): New York City Agar – VCAT (Amphoteracin B) o
Antibiotics: V ancomycin = inihibits Gram (+) C olistin = inihibits Gram (+) bacilli N ystatin = inhibits Yeasts T rimetophrim = inhibits swarming of Proteus 3.
OXIDASE TEST / TAXO N – Presumptive Test Procedure: Rub the colony into a filter paper. (+) PURPLE The reagent is already incorporated into the filter paper or the reagent is placed on the colony itself. Reagent = 1% tetramethyl-p-phenylenediaminedihydrochloride Positive Control: Pseudomonas (easily grows) (+): Neisseria, Moraxella, Aeromonas, Pseudomonas
4.
CARBOHYDRATE FERMENTATION TEST – Confirmatory Test Media: CTA (Cysteine Trypticase Agar + Phenol Red) Phenol Red – indicator detecting acid in this test No need for CO2 requirement! CO2 is only for culture. Fermentation of GLUCOSE only: (+) YELLOW
5.
6.
SUPEROXOL CATALASE TEST Reagent = 30% H2O2 (+) Neisseria gonorrhea BETA LACTAMASE TEST: (+) Color Change Performed when Penicillin resistant. Held on Primary Culture (since Plasmid is lost during subculture) a. Chromogenic Cephalosporin Test: (+) Pink/Red b. Iodometric Test – Iodine and Pen: (+) Colorless Acidimetric Test Phenol Red and Pen: (+) Yellow
B. Neisseria meningitides - Gram negative diplococci that produces mucoid colony in culture media Carrier: nasopharynx Virulence: Capsule and enotoxin (N. gonorrhoeae – pili) Diseases: Meningitis, Meningococcemia Waterhouse Freiderichsen – hemorrhage of adrenal gland Specimen: Blood and CSF Serotypes: A, B, C, Y, W135 (Capsular Ags) Laboratory Diagnosis: 1. Gram Stain 2. Culture: CAP (+); BAP (+) 3. Oxidase Test (+) Purple 4. Carbohydrate Fermentation – Glucose and MALTOSE C. Moraxella catarrhalis/Branhamella Oxidase Test (+) Reduce NO 3 to NO 2 DNAse (+) (Best); M. lacunata and M. denitrificans = DNAse (-) “Hucky Puck Colony ” rd 3 cause of otitis media Lab Diagnosis: Butyrate Esterase Disc Test (Tributyrin Hydrolysis) = (+) Blue Co lor Assacharolytic (not degrading any sugar) ; Beta Lactamase Producer : Penicillin resistant Also grows in Nutrient Agar.
N. gonorrheae N. meningitidis
Oxidase + +
M. catarrhalis
+
Sugar/CHO Glucose Glucose Maltose None
DNAse -
TMA + +
+
+
Carbohydrate Fermentation Test N. meningitides N. gonorrheae N. secca N. lactamica M. catarrhalis NOTE:
GLU + + + + -
MAL + + + -
LAC + -
SUC + -
ACID FAST ORGANISMS “Acid Fast” = Acid alcohol resistant not decolorized by acid alcohol retaining the RED color of CARBOLFUCHSIN due to the presence of MYCOLIC ACID (a long chain of fatty acid that makes Mycobacteria the bacteria with the highest amount of lipid)
I. MYCOBACTERIA ( Mycobacteria = “Mataba at Mabagal”)
ACID FAST BACILLI (mycolic acid) Growth: 2-3 weeks 8 weeks before reporting as NEGATIVE Slow growers (except M. fortuitum, M. chelonei) = 1 week only “Much granules”; aerobic non sporeforemer; non motile
3 GROUPS: A. MYCOBACTERIUM TUBERCOLISIS COMPLEX - causes TB 1. M. tuberculosis- pulmonary TB 2. M. bovis- intestinal tuberculosis (BCG) (Acquired through drinking unpasteurized milk) 3. M. africanum- pulmonary TB (Africa) B.
MOTT (Mycobacteria Other Than Tuberculosis / Non Tuberculosis Mycobacteria) can cause pneumonia but not tuberculosis
C.
MYCOBACTERIUM LEPRAE cannot be grown on agar obligate intracellular
Decontamination-Digestion performed only on sputum (since it is not sterile), therefore CSF does not need this process anymore since it is a sterile specimen. a. NALC-NaOH (2-4%) + Na citrate (GOLD STANDARD) Na Citrate – removes the metallic compound b. Zephiram Trisodium PO4 c. 6% Oxalic Acid – used when the specimen is contaminated by Pseudomonas such as urine and stool sample d. Dithiothreitol (sputulysin) – lyses the sputum and the Acid Fast Stain
2.
Acid Fast Stain AFB GRADING NATIONAL STANDARD No AFB / 300 fields 0 1-9 AFB / 100 fields +n 10-99 AFB / 100 fields 1+ 1-10 AFB / field in at least 50 fields 2+ >10 AFB / field in at least 20 fields 3+ Note: Spot-Morning-Spot – requires 1 morning sputum specimen used to view Acid Fas Organisms by DSSM (Direct Sputum Smear Microscopy) Ideal Size of Sputum Smear = 2x3cm 300 Fields = 2 Lines (should be read before declaring 0 or No AFB)
150 Fields 150 Fields
3. A.
MYCOBACTERIUM TUBERCULOSIS (Koch Bacillus) Obligate aerobe requiring slight opening of LJ Media to allow O 2 to enter; require 5-10% CO2 for growth Cauliflower, Buff-colored (non-photochromogen) colonies in LJ Virulence: Cord factor (causes sticking of mycobacteria, producing cording effect on Acid Fast Stain) Grading System (3+) Sulfatides Laboratory Diagnosis: 1. Gram Stain - qualify specimen (accept or reject specimen) a. Sputum: <10 Epithelial Cells; > 25 PMN b. Saliva: >10 Epithelial Cells; <25 PMN
Skin test
4.
Sensitive and simplest test for cell-mediated immunity (Type IV) Ex. Tuberculin Test
Culture – still needed even if (+) in Gram Stain. Remember that when you do culture, you should also do susceptibility test since there are drugresistant mycobacteria. Note: Type of media for susceptibility test for mycobacteria: Agar Based (Middlebrook 7H11) Type of media of the best culture media for mycobacteria: Egg Based (LJ)
A.
AGAR BASED MEDIA a. Middlebrook 7H11 - (for AST) clear media used for susceptibility testing Wrinkled colony of MTB Duboi’s Oleic Acid Medium b. Mitchison’s Medium
B.
EGG BASED MEDIA: Malachite Green Type of sterilization: INSPISSATION a. Lowenstein Jensen Medium (BEST) Malachite Green serves as an INHIBITORY AGENT since sputum is non-sterile Glycerol : serves as the CARBON SOURCE for LJ Take note that Nocardia can also grow in LJ other than Mycobacteria spp. Cauliflower, Buff-colored (yellow brown) colonies in LJ b. Petragnani Medium c. American Thoracic Society Medium d. Dorset Egg Medium
C.
5.
LIQUID MEDIA For RAPID CULTURE SYSTEM a. Bactec 12B b. Septi-Chek c. Middlebrook 7H9
Biochemical Tests for Mycobacteria a.
NIACIN TEST Requires colony coming from the LJ media PPL: Niacin + Niacin Ribonucleotide + Aniline Dye + Cyanogen Bromide = YELLOW (+) (+) M. tuberculosis (yellow) (-) M. bovis
b.
NITRATE REDUCTION TEST Reagents: a. HCl b. Sulfanilamide c. N-napthtylethylene diamine (+) M. tuberculosis (pink/red) (-) M. bovis
c.
TCH SUSCEPTIBILITY TEST
B.
d.
CATALASE TEST at 68’C (Heat Stable Catalase) Medium: Tween 80 Reagent: 30% H2O2 PPL:Tween 80 + Mycobacteria + 30% H2O2 + Heat at 68’C Result: >45mm height of gas bubbles (+) M. kansasii, M. avium (-) M. tuberculosis
e.
TWEEN 80 HYDROLYSIS TEST Patient: Tween 80 HOH of Tween 80 (+) M. kansasii (red) (-) M. avium
f.
TELLURITE REDUCTION TEST Patient: Telurite Black Metallic Tellurium (+) M. avium (blackening of media) (-) M. kansasii
g.
ARYSULFATE TEST Tripotassium phenolphthalein disulfide / Sulfate acted upon by Arylsulfatase to produce Free Phenolphthalein (+) M. fortuitum-chelonei (pink/red)
MOTT (ATYPICAL MYCOBACTERIA)- NTM A. Photochromogens - ROUNYOUN’S I = YELLOW 1. M. kansasii 2. M. marinum 3. M. asiaticum, M. simiae B. Scotochromogens - ROUNYOUN’S II = ORANGE/YELLOW 1. M. scrofulaceum (scrofula) 2. M. szulgai 3. M. gordonae (Tap Water Bacillus) C. Non photochromogens - ROUNYOUN’S III = CREAM/BUFF COLORED 1. M. avium (#1 NTM) or 2. M. intracellulare (Battery Bacillus) 3. M. ulcerans (Buruli) 4. M. xenopi (Hot and Cold Water T aps) 5. M. triviale, M. haemophilum 6. M. malmoense, M. terrae, M. gastri D. Rapid Growers- ROUNYOUN’S IV (<7days) 1. M. fortuitum 2. M. chelonei 3. M. phlei- provide CO2 4. M. smegmatis- confused with MTB in urine (formerly known as M. lacticola)
PHOTOCHROMOGENS
TWEEN 80 HYDROLYSIS (+) M. kansasii M. marinum M. asiaticum I NITRATE REDUCTION
(+) M. kansasii
NON PHOTOCHROMOGENS (Mycobacterium terrae Complex - MTC)
(-) M. simiae
CATALASE (>45mm) (+) M. terrae Complex M. triviale I 5% NaCl
(-) M. marinum (+UREA) M. asiaticum (+) M. triviale
(-) M. avium Complex M. xenopi
(-) M. terrae Complex
SCOTOCHROMOGENS
NITRATE REDUCTION (+) M. szugai
(-) M. scrofulaceum M. gordonae I UREASE (+) M. scrofulaceum
(-) M. gordonae
NON PHOTOCHROMOGENS (Mycobacterium avium Complex - MAC)
UREASE (+) M. avium M. intracellulare (MAC)
(-) M. xenopi
RAPID GROWERS
ARYLSULFATASE (+) M. fortuitum – chelona I NITRATE REDUCTION 5%NaCl IRON UPTAKE
(+) M. fortuitum
(-) M. chelonae
(-) M. smegmatis
ROUNYOUN’S I
ROUNYOUN’S II
ROUNYOUN’S III M. avium complex - differentiated by Nucleic Acid Amplification Test/PCR
ROUNYOUN’S IV Both: Arylsulfatase (+) Grows on MacConkey w/o Crystal Violet
MOTT SUMMARY OF DIFFERENTIATION PHOTOCHROMOGENS YELLOW M. kansasii Chronic Pulmonary “Yellow Bacillus” Disease Nitrate (+) (Pneumonia) Swimming Pool Nitrate(-) M. marinum Granuloma Grows well at 30’C SCOTOCHROMOGENS ORANGE / YELLOW Scrofula agent Urease (+) M. scrofulaceum (Cervical Tween 80 (-) Lymphadenitis) M. gordonae (Non Pathogenic) Urease (-) “Tap Water Bacillus” Tween 80(+) NON CREAM / BUFF COLORED PHOTOCHROMOGENS Chronic Pulmonary M. avium Catalase (+) Disease (among Tellurite (+) AIDS patients) M. intracellulare (Battery Bacillus) M. xenopi (Hot and Cold Water Taps) RAPID GROWERS M. fortuitum
M. chelonei
Wound Infection
Grows at 4’C and 45’C
GROWS <7 DAYS All Positive Differentiated by: Nitrate Reduction All Negative 5% NaCl Iron Uptake
Photoreactivity/Photosensitivity Uses 2 LJ media (light and dark tube) a. Light Tube – without alumunim foil b. Dark Tube – with aluminum foil Remove the aluminum foil in the dark tube when there is growth on the light tube, and simultaneously check for the growth of the 2 tubes.
AUTOMATED TEST FOR MYCOBACTERIUM 1. Bactec 460 Middlebrook 7H12 (RIA) 14 14 PPL: C Palmitic Acid + Organisms = CO2 (measured) POSITIVE Result: More than 10 growth index
C.
2.
Mycobacteria Growth Indicator Tube (MGIT) Fluorometric Based (less harmful) POSITIVE Result: Fluorescence PPL: Increased bacteria = Increased O 2 Consumption = Fluorescence
3.
Bactec 12B + NAP (Growth Inhibition Test) p-nitro acteylamino beta NAP - inhibits M. tuberculosis (Sensitive) POSITIVE Result: No growth
MYCOBACTERIUM LEPRAE (Hansen’s Bacillus) AFB, “cigarette-packet / picket fence ” Obligate Intracellular Not cultivated in agar (in vitro) Hydrolize DOPA Tropism to peripheral nerves
Disease: Leprosy (Hansen’s Disease) Lepromine Test – skin test for leprosy a. Lepromatous: Lepromine (-), many AFB; characterized by LEONINE FACE 2 Types of Reaction: 1. Fernandez = Early 2. Mitsuda = Late b. Tubercoloid Lepromine (+), few AFB; good prognosis than Lepromatous Laboratory Diagnosis 1. Clinical Findings- basis of diagnosis 2. Culture - foot pads of armadillo (since their feet are cold, t. leprae like cold environment) 3. Fite Faraco Stain Treat: Dapsone OTHER MYCOBACTERIA: 1. M. genavensi- disseminated infections in AIDS; BACTEC (+) 2. M. paratuberculosis- Crohn’s Disease 3. Rhodococcus equi - pleomorphic (rod cocci / vice versa) every 24 hours, and pink colonies (+); CAMP Test (+) with S. aureus beta hemolytic
8.
II. NOCARDIA
Partially acid fast (1% H2SO4) (Modified Acid Fast) Urease (+) Gram (+)branching rod FUNGUS LIKE BACTERIA (Just like Actinomyces) Cause: PNEUMONIA (Sputum) Best Media: Casein Medium Also grows in LJ Media. To differentiate with Mycobacteria = GRAM STAIN. Nocardia is fungus like. Differentiate Species by: CASEIN HYDROLYSIS N. asteroids (-) o N. brasiliensis (+) o
III. CORYNEBACTERIA
1.
Pleomorphic Gram (+ )rods Has similarity with Listeria. Arrangement: Clubshape, X, Y, V L, chinese letters o Palisade appearance (side by side) o Babes-Ernst (metachromatic granules) Normal Flora: Oral Cavity, Skin, Genital (just like Candida albicans) Non motile, No spore, capsule BAP: raised, transluscent gray colonies Catalase (+)
Corynebacterium diphtheriae (Kleb Loeffler’s Bacillus) Virulence factor: Exotoxin Labile A,B Disease: Diphtheria (grayish pseudomembrane on tonsils and pharynx (Upper respiratory infection) Specimen: Throat Swab (Pharyngitis or Diptheria) if Diptheria, culture in Loeff ler’s Medium to see the Metachromatic Granules Lab Diagnosis: 1. LAMB – Metachromatic granules (Pai Loeffler) 2. Tellurite Media: Gray to black colony (since it can reduce Tellurite) 3. Urease (-): (Presumptive test) The only pathogenic corynebacteria which is Urease (-) 4. CHO ferment: dextrose & maltose (+) 5. ELEK test: definitive test (confirmatory test) 6. Schick’s test – skin test for diphtheria 7. Culture similar to C. pseudotuberculosis, C. ulcerans . (Arthrobacter culture similar to Brevibacterium)
Culture on : a. Blood Agar (White and dry colony) b. Tinsdale (black colony w/ brown halo) c. Potassium tellurite (gray to black colony), d. Loeffler’s serum agar e. Pai coagulated egg f. Clauberg Mclead g. Cystine Tellurite BAP (CT-BAP) ( gun metal gray colony) – media used to differentiate the biotypes of C. diptheriae based on the size of their colonies *Potassium tellurite : inhibits normal flora
BIOTYPE OF C. diphtheria (CT-BAP) 1. C. gravis: gray, large, Beta hemolytic, starch/glycogen fermentation (+) 2. C. intermidius: black, small, non hemolytic 3. C. mitis : medium-size, black, beta hemolytic, starch/glycogen (-) “PATHOGENS” - All ferment DEXTROSE and MALTOSE C. diphtheriae C. ulcerans C. pseudotuberculosis
Urease
Nitrate red.
Starch HOH
( –) + +
+
– + –
+/-
Note:
C. diphtheria is the only human pathogen C. ulcerans and C. pseudotuberculosis are animal pathogens C. ulcerans = mastitis in cattle o o C. psedutuberculosis = TB like infection in animals Starch Hydrolysis used to differentiate C. ulcerans and C. pseudotuberculosis (+) Disappearance of Blue color (C. ulcerans) Reagent: Iodine
“DIPTHEROIDS” (Normal Flora) C. xerosis C. pseudodiphthericum (Hoffman’s bacillus) C. jeikeium
Urease –
Nitrate red
+
CHO ferment Glucose, Maltose, Sucrose None
+
Glucose
–
+
+
Note:
Glucose = Dextrose Sucrose = Saccharose All of them are normal flora but can cause ENDOCARDITIS Specimen: Blood o C. xerosis – causes conjunctivitis C. pseudodiptheriticum – oral flora C. jeikeium – common for patients with HIV ; cause of Prosthetic Valve Endocarditis (like S. epidermidis); drug resistant (beta lactamase producer) C. urealyticum – Urease (+), causes UTI
2.
Corynebacterium minutissimum agent of erythrasma Diagnosis: “coral red fluorescence” on wood’s lamp (skin infection) Red Fluorescence due to PORPHYRIN.
3.
Arcanobacterium haemolyticum Beta hemolysis on SBAP, lipase and lecithinase(+) (+) reverse CAMP with S. aureus (+) Inverted Triangle / Triangle Type of Hemolysis
Note: CAMP (S. aureus) = S. agalactiae, R. equi Reverse CAMP (S. aureus) = Arcano Reverse CAMP (S. agalactiae) = C. perfringens
GRAM POSITIVE BACILLI Gram Stain
LACTOBACILLUS Gram (+) Rod
SPORES (-) Corynebacterium Listeria Lactobacillus Actinomyces Kythria Erysiphelothrix I CATALASE TEST
(+) Bacillus Clostridium
O2 Sulfur Granules Acid Catalase
O2 Catalase Gas
Catalase Oxidase Motility Growth at 4’C Esculin VP (Acetoin) Methyl Red
Catalase O2 AF Urease
+ – –
BACILLUS 1.
(+) Corynebacterium Listeria
Anaerobic (Aerotolerant) – + –
ACTINOMYCES Gram (+) Branching Rod Anaerobic
(-) Lactobacillus Actinomyces Kuthria Erysiphelothrix
BACILLUS Aerobic + –
CLOSTRIDIUM Anaerobic – + (Foul Odor)
CORYNEBACTERIUM + – – – – – +
LISTERIA + – + (22’C) + + + -
NOCARDIA + Aerobic AFO +
ACTINOMYCES – Anaerobic NAFO –
Bacillus anthracis (Anthrax Bacillus) Gram (+) Rods, chain – Bamboo shape appearance Largest bacteria (Smallest is Mycoplasma) Non motile, spore forming, zoonotic (acquired from animal source) Virulence factor: Exotoxin (edema and lethal) o Capsule o made up of Glutamic Acid (D Glutamate) BICARBONATE MEDIUM (Enhances the capsule formation of B. anthracis) McFadyean’s Reaction – presumptive test for the presence of Capsule (+) Pink = Capsule; Blue = Bacilli (due to Methylene Blue) Disease: 1. Malignant pustule – black eschar 2. Woolsorter’s / Rag Picker’s Disease - pulmonary anthrax 3. Gastroenteritis – bloody diarrhea (intestinal anthrax) Lab Diagnosis: 1. Selective Medium: PLET (Contains antibiotic: POLYMYXIN from its name Polymyxin Lysozyme EDTA Thalous Acetate) 2. COLONY a. Medusa Head Colony b. Lion Head Colony c. Serrated Irregular Colony d. Inverted Pine tree (Grows on GELATIN TUBE MEDIA) 3. STRING OF PEARL TEST (0.05 units PEN) – BAP (Microscopically seen) 4. ASCOLI TEST = serologic precipitation test: (+) Precipitation Ring 5. Serologic Tests: PCR – best method for diagnosis of anthrax Fluororescence Ab test ELISA
Note: Summary of Tests for some organisms: Ascoli = B. anthracis Anton = Listeria Casein = Nocardia Nagler = C. perfringens
CLOSTRIDIUM
Note:
Inverted Pine Tree = B. anthracis Umbrella = L. monocytogenes Pipe Cleaner/Test Tube Brush = Erysiphelothrix
Obligate anaerobe gram (+), endospore Habitat: human and animal Saccharolytic (fermentation since anaerobic process) except: C. tetani, C. septicum
3 Types pf Clostridium 1. Neurotoxic: C. tetani and C. botulinum (more dangerous since they act on CNS) 2. Histotoxic: C. perfringens and C. septicum 3. Eneteric: C. difficile
1. 2.
Bacillus cereus ( Fried Rice Bacillus/Spore Rice Grain) Virulence: Exotoxin (Cholera like T)
Motility Capsule Hemolysis Growth 45’C Salicin Ferment Penicillin G Gelatin Hydrolysis & PEA
B. anthracis – + Non Hemolytic – – S –
B. cereus + – Beta Hemolytic + + R +
Note: Once a bacteria has CAPSULE = NON MOTILE!
3.
4.
Bacillus subtilis Gram (+) Rod in chain, central spore Common laboratory contaminant Source of antibiotics Eye infection in heroin addict Used as QC in OVEN BAP – large, flat dull, beta hemolytic, ground glass Has similarity with P. aeruginosa Difference: P. aeruginosa (moist colony); B. subtilis (dry colony) Bacillus stearothermophilus Flat sour spoilage; acid without gas; QC for AUTOCLAVE Note: Flat sour spoilage = B. stearothermophilus Bloated sour spoilage = C. botulinum
Clostridium perfringens (C. welchii) The only ENCAPSULATED clostridium (The only Non mot ile) Double Hemolysis “Box car shape bacillus” SUBTERMINAL SPORE Source: wound contamination with soil Diseases: Gas gangrene, myonecrosis (skin infection) Food poisoning, enterotoxins Necrotic enteritis Lab Diagnosis: 1. CHOPPED MEAT – growth + gas (anaerobic growth) anaerobic broth. Promotes spore formation Growth: Turbidity 48 hours growth of anaerobes 2.
BAP – Target or Double Zone of Hemolysis (Exclusive for C. perfringens) Alpha Hemolysis (Partial/Incomplete) (ALPHA TOXIN) Beta Hemolysis(Complete) (THETA TOXIN)
3.
NAGLER TEST – Lecithinase Test (Alpha Toxin) Due to alpha, lecithinase C, phospholipase C Media: Mc Clung or Neomycin Egg Yolk – medium to demonstrate lecithinase (+) OPALESCENCE on agar without anti toxin
4.
REVERSE CAMP TEST – uses S. agalactiae (Group B Strep) (+) Arrow Head Zone of Beta Hemolysis
Note:
5.
STORMY MILK FERMENTATION (+) COAGULATE CASEIN/ Clotting of protein
Note: Swarmers! Proteus (Gram – ) C. tetani (Gram + ) C. septicum (Gram +)
Note: CAMP (S. aureus) = S. agalactiae, R. equi Reverse CAMP (S. aureus) = Arcano Reverse CAMP (S. agalactiae) = C. perfringens 2.
Flaccid Paralysis – C. botulinum (Nanlalambot) Spastic Paralysis – C. tetani (Naninigas)
4.
Clostridium botulinum (Canned Good Bacillus) Virulence: Toxin heat labile – block release of acetylcholine ( flaccid paralysis; Virus = Poliovirus) Botulinum Toxin (neurotoxin) most potent toxin in human, acts on CNS used as BOTOX Diseases: (Not Cultured) Wound Botulism – spore on wound Infant botulism – honey bee (floppy baby) Botulism = most sever food poisoning
Clostridium difficile Colon flora Antibiotic (clindamycin) associated pseudomembranous enterocolitis (violent diarrhea) Lab Diagnosis Direct detection of toxin (stool) Toxin detection by EIA Cytotoxin assay Culture: CCFA – yellow (horse manure) Indicator: Phenol Red
Note: Food Poisoning (Wag mag aral sa CSB, maraming nafofood poison!) C. perfringens/ C.tetani S. aureus B. cereus 3.
Clostridium tetani (Tackhead Bacillus) TERMINAL SPORE(tennis racket, drumstick) Asaccharolytic Swarming! Has special affinity to Iron. Virulence: Exotoxin (tetanolysin and tetanospasmin) binds to ganglioside receptors and inhibit neurons in CNS – spastic paralysis Lock Jaw, Risus sardonicus (Sardonic Smile), Opisthotonus (Arching of back), Neonatal Tetanus Lab Diagnosis: Clinical findings – basis of diagnosis Terminal spore = OVAL TERMINAL SPORE Terminal Spore
Fermentation of Glucose
Gelatin
ANAEROBIC BACTERIOLOGY 5% CO2 + 10H2 + 85% N2 FOUL ODOR Growth: 48 hours Collection: a. needle aspiration b. suprapubic aspiration Media: 1. Chopped meat 2. Thioglycollate - aerobic and anearobic 3. Reduced media reduced oxygen anaerobic BAP, Schaedlar, Bacteroides bile esculin 4. Laked kanamcin vancomycin BAP (LKVBA) 5. Anaerobic PEA, egg yolk agar
Methods to Promote Anaerobiosis: Anaerobic Jars Gas Pak Jar (Anaerobic Jar) o Incubation Time: 48 Hours Gas Pak Envelope = CO 2 + H (Enclosed) When opened: O 2 (that enters) react with H H2O (Moisture): Indicator that the O2 has been removed Mcintosh Fildes Jar o Torbal o Brewer o
Note: Candle Jar is not anaerobic jar! It cannot be used for anaerobic organisms. Chopped Meat anaerobic broth. Promotes spore formation Growth: Turbidity Thioglycollate medium Indicator: Resazurin (Pink). Seen at the surface, once it goes down, indicates presence of O2. Note: When exposed to O2, there will be no growth. In case of exposure, BOIL or AUTOCLAVE to remove O2 Stored at: Dark Room at Room Temp PRAS – Pre-Reduced Anaerobically Sterilized (Method: Roll Tube of Hungate)
Indicators of Anaerobiosis: a. Methlyene Blue: Blue Colorless (O 2 has been removed) b. Resazurin: Pink Colorless (O 2 has been removed)
Identification: Kanamycin Vancomycin Colistin (KVC) presumptive test to Identify anaerobic bacteria B. fragilis is the most important anaerobic bacteria since it a beta lactamase producer (penicillin resistant), which is seen in the GIT (normal flora). Also requires 20% Bile for growth (Bile Resistant) Porphyromonas is Vancomycin (S); (Bile Sensitive) Prevotella is Vancomycin (R); (Bile Sensitive) Characteristics of Anaerobes Brick red fluorescence Red fluorescence Pitting of agar Double zone hemolysis Swarming Molar tooth colony, sulphur granules Breadcrumb colony
Prevotella, Porphyromonas Veillonella B. ureolyticus C. perfringes C. tetani, C. septicum (Gram +) Actinomyces israelii F. nucleatum
A. GRAM (+) ANAEROBIC BACILLI 1. Actinomyces – fungus-like bacteria - Catalase (-) Actinomyces bovis – lumpy jaw Actinomyces israeli – draining sinus tract with sulfur granule; Molar Tooth Colony 2. Bifidobacterium dentium – Oral and GIT (Causing Periodontitis) 3. Eubacterium lentum - Oral and GIT (Causing Periodontitis) 4. Propionebacterium acne (Formerly known as Corynebacterium acne) anaerobic diptheroid skin flora; blood culture contaminant Catalase (+) and Indole (+) 5. Lactobacillus GIT and Vaginal Flora Increased during Child bearing years Inhibits Gardnerella vaginalis; Promotes Candidiasis Catalase (-) 6. Mobiluncus – motile organism causing vaginitis (G. vaginitis)
B. GRAM ( –) ANAEROBIC BACILLI (GIT FLORA) 1. Bacteroides fragilis (Colon Bacillus) needs 20% bile (Bile resistant) Specimen: peritoneal fluid, instestinal abscess Black Colony on Bacteroides Bile Esculin Medium Catalase (+), Indole (-) All Bacteroides are Indole (-) except: Bacteroides Thetaiotamicrons 2. Porphyromonas and Prevotella: a. Porphyromonas asaccharolytica – black pigment, red fluorescence on UVL, Van (S) b. Prevotella melaninogenica – black pigment, red fluorescence on UVL, Van (R) 3. Fusobacterium Catalase (-), Indole (+) a. F. nucleatum – breadcrumb colonies; fusiform rod (spindle shaped) b. F. necrophorum – Synergistic effect with Borrelia vincentii ( a spirochete which is a trench mount agent ) causing Vincent’s angina (giginvitis/gum disease) 4. Bacteroides ureolyticus – pitting of agar
C. GRAM (+) ANAEROBIC COCCI 1. Peptostreptococcus anaerobius – SPS sensitive , Catalase (-) indole (-) 2. Peptostreptococcus asaccharolyticus - Catalase (-), indole (-) 3. Peptococcus niger (Staph-like) - Catalase (+)
Propionebacterium Bacteroides spp. Bacteroides thetaiotamicrons Fusobacterium spp. Peptostreptococcus anaerobius Peptostreptococcus asaccharolyticus Peptococcus niger
CATALASE + + + +
INDOLE + + -
2.
3.
NITRATE REDUCTION TEST(Nitrate Broth) PPL: NO3 → NO2 Reagent: sulfanilic acid and a-napthylamine = (+) RED COLOR If (-) COLORLESS: add ZINC DUST/POWDER to detect the presence of unreduced nitrate Turns colorless: positive o Turns red: negative (unreduced nitrate) o
NITRITE REDUCTION TEST PPL: NO2 → N2 gas (DURHAM TUBE – Inverted Test tube f or Gas Production) Reagent: sulfanilic acid, a-napthylamine + zinc dust (+) COLORLESS (A. faecalis) (-) RED (A. piechaudii)
NOTE: H2O2 for the Catalase Test for Anaerobic Bacteria = 15% H2O2 (not 3%)
4.
ONPG TEST ONPG via B-galactosidase to Orthonitrophenol B-galactosidase degrades Lactose to Galactose Rapid Lactose Fermenter – ONPG (+); B-galactosidase + Permease o Late Lactose Fermenter – ONPG (+); B-galactosidase only o Non Lactose Fermenter – ONPG (-) o Note: Lactose Fermentation Test: Pathogenicity test for Enterobacteriaceae (Where NON LACTOSE are mostly pathogenic) (+) yellow (E.coli) (-) change in color (S. typh imurium)
D. GRAM (-) ANAEROBIC COCCI 1. Veillonella parvula – fluoresce red UVL; oral flora; nitrate (+) 2. Megasphera 3. Acidaaminococcus
DEFINITIVE Test for Anaerobic Bacteria: GLC/HPLC (detects acid)
Diagnostic test for GRAM (-) BACILLI: Note: Growth in MacConkey Agar indicates growth of Gram (-) Bacilli. BUT some Gram (-) Bacilli does not grow in MacConkey. The following are few examples: a. Haemophilus – requires X and V Factor (not in Mac) b. Brucella/Legionella – needs special growth factor (most bacteria ending with –ella does not grow in Mac, except for Salmonella) 1.
OXIDASE TEST (Presumptive Test for Gram Neg Bacilli) Cytochrome oxidase (indophenol blue) (+) P. aeruginosa (Bluish Purple) (-) E. coli
Note: Oxidase test: Presumptive Test for Gram – Bacilli Oxidative-Fermentative Test: Presumptive Test for Non Fermentative Organism
5.
LOA TEST / AMINO ACID ENZYME TEST (+) purple (-) yellow 3 Enzymes: 1. Decarboxylase (anaerobic): Lysine to Cadaverine 2. Dehydrolase (anaerobic): Ornithine to Putrescine 3. Deaminase (aerobic – Slant): Arginine to Citrulline “De” – removal of amino group MEDIUM; Moeller’s Decarboxylase Medium Indicator: Bromceresol Purple o MINERAL OIL: Prevents the entry of O 2 Total of 4 Test Tubes (with 1 being the Control) (+) Lysine Decarboxylase: K.pneumoniae (-) Lysince Decarboxylase: E.cloacae
6.
TRIPLE SUGAR IRON (TSI)
7.
Glucose + Lactose + Sucrose + Iron (Reacted by H 2S) G:L:S Ratio (1: 10: 10) o pH indicators: a. Phenol Red = red to yellow (Acid Production) b. Ferrous Sulfate (H2S Production) = black TSI Reaction:
ACID SLANT
YELLOW
A/A ACID BUTT
ALKALINE SLANT
YELLOW RED
K/A
K/K
Lactose Sucrose Glucose
Glucose ACID BUTT
YELLOW
ALKALINE SLANT ALKALINE BUTT
RED None
(+)Blackening due to Ferrous Sulfate
Gas
(+)Splits Medium (Space)
KIA – Kligler’s Iron Agar (G -L-S Iron) RDA – Russell’s Double Agar (G -L)
Indicators: a. Bromcresol Purple b. Ferric NH4 Citrae (H2S Production) = black
Lactose Fermenter Hafnia Arizona Citrobacter E. coli Enterobacter Klebsiella Non Lactose Fermenter P-M-P Salmonella Shigella Serratia Yersinia Non Fermentative Organism Pseudomonas
RED
H2S
LYSINE IRON AGAR (LIA) Lysine deamination – slant (aerobic) Lysine decarboxylation – butt (anaerobic)
H2S Producer Salmonella Proteus Aerogenic Organisms
K/K
NEG (-) POS (+)
LYSINE DEAMINASE
K/A
NEG (-) NEG (-)
LYSINE DEAMINASE
R/A
POS (+) NEG (-)
LYSINE DEAMINASE
H2S
(+)Blackening due to Ferric Ammonium Citrate
LYSINE DECARBOXYLASE
LYSINE DECARBOXYLASE
LYSINE DECARBOXYLASE
PURPLE PURPLE PURPLE YELLOW RED YELLOW
Note: Summary of Some Indicators on Agars and Tests: 1. Phenol Red a. TSI b. XLD c. MSA d. Christensen’s Urea e. BGA 2.
Bromthymol Blue a. HEA b. SCA c. TCBS d. Utilization Tests (Citrate, Acetate, Acetamide, Malonate)
3.
Neutral Red a. MacConkey b. SSA c. DCA
4.
Bromcresol Purple = LIA
DESCRIPTION
8.INDOLE TEST
9. RAPID SPOT INDOLE TEST
Indole from a. Amino Acid: Tryptophan b. Enzyme: Tryptophanase
REAGENT
MEDIA
Kovac’s/Erlich’s Reagent = PDAB
SIM H2S Indicator: Lead Acetate
INDICATOR
POSITIVE
NEGATIVE
(+)RED
(-) YELLOW
POSITVE
NEGATIVE
(Anaerobic Bacteria)
Filter paper strips impregnated with PDAB
(+)BLUE
Screening for indole production.
10. METHYL RED TEST (Opposite VP)
11. VOGUES PROSKAUER TEST
12. CITRATE UTILIZATION TEST 13. ACETATE UTILIZATION TEST 14. ACETAMIDE UTILIZATION TEST
MRVP (broth) (Aka: “Clark And Lubs Dextrose”)
Mixed acid of glucose fermentation
PRODUCT: Acetoin or acetylmethylcarbinol
COBLENTZ METHOD: A-napthol and 40% KOH in creatine (GRAM POS COCCI – Strep. mutans) Utilize Citrate as source of carbon and energy Acetate → alkaline pH (blue) Acetate → acid pH (green)
Simmon’s Citrate Agar
Acetamide → NH3 (BLUE)
Product: (NH4)2CO3 (Alkaline Ammonium Carbonate)
(+)RED
(-) YELLOW
BARRITS METHOD: A-napthol and 40% KOH (GRAM NEG BACILLI – E. cloacae)
15. MALONATE UTILIZATION TEST
16. UREA HYDROLYSIS TEST
Methyl Red
Christensen’s Urea
Bromthymol Blue
(+)RED
(-) YELLOW
(+)BLUE
(-) GREEN
Bromthymol Blue
(+)BLUE
(-) GREEN
Bromthymol Blue
(+)BLUE
(-) GREEN
Bromthymol Blue
(+)BLUE
(-) GREEN
Phenol Red
(+)RED/PINK
(-) YELLOW
(pH below 4.4); (E.coli)
E. cloacae
(KESH) Klebsiella, Enterobacter Serratia Hafnia
(P. aeruginosa)
(E. coli)
(E. coli)
(Shig. flexneri)
(P. aeruginosa)
(S. maltophilia)
(KECH) Klebsiella, Enterobacter Citrobacter Hafnia
17. PHENYLALANINE DEAMINASE (PAD)
ENTERIC MEDIA (Selective and Differential)
Organism is found at the slant since it is an aerobic enzyme
Presumptive test for Proteus, Morganella, Providencia
18.
KCN BROTH TEST (+) turbid (growth of organism) o
Klebsiella
o
Enterobacter
o
Proteus/Providencia
o
Serratia
o
Citrobacter
(-) clear ( Salmonella )
Medium EMB
Mac XLD
Inhibitory Eosin Y Methylene blue CV, bile salt Bile salt
HEA
Bile salt
Salicin Lactose Sucrose
Bromthymol blue
DCA SSA
Bile salt Bile salt
Lactose Lactose
Neutral red Neutral red
BSA
Glucose
TCBS
Brilliant green Bile salt
Brilliant Green A.
Brilliant Green
Bismuth sulphite Bromthymol blue Phenol Red
19. STRING TEST
ID of V. cholerae
Reagent: 0.5% Na deoxycholate
(+) String Like (V. cholerae)
CHO Lactose
Lactose Xylose Lactose Sucrose
Indicator Eosin Y Methylene blue Neutral red Phenol red
20. ESCULIN HYDROLYSIS TEST
(+) Black (K. pneumoniae)
(-) Yellow (S. flexneri)
21. MUG TEST (UVL)
(+) Blue Fluorescence = E.coli (exceot. E. coli 0157 H7)
(-) No Fluorescence = P. aeruginosa
Sucrose
22. GELATIN HYDROLISIS TEST
(+) Gel Liquifies = P. vulgaris
(-) Gel Solidifies ; E. aerogenes
LF Red/purple
NLF Colorless
Red/pink Yellow (E. coli)
Colorless Red (Shigella or NLF) Black (Salmonella) Yellow Green (Shigella or NLF) Black (Salmonella) Red/pink Colorless Red Colorless (E. coli) (Shigella or NLF) Black (Salmonella) Black colony – Salmonella typhi Yellow Green (Vibrio) Black colony - For other Salmonella spp not S. typhi
Note:
All of the above inhibits the growth of GRAM (+), promoting the growth of GRAM () BSA and TCBS are the only m edium above that do not have Lactose. BSA cannot differentiate LF from NLF, only selective to Salmonella typhi
Note: Summary of H2S Indicators: 1. TSI, and BSA = Ferrous Sulfate 2. LIA, XLD and HEA = Ferric NH4 Citrate 3. SIM = Lead Acetate 4. SSA = Ferric Citrate
ENTEROBACTERIACEAE
All are glucose fermenters (A/A or K/A only). K/K (no sugar fermented) is not possible. All are motile (peritrichous flagella) except Klebsiella (capsulated) and Shigella (noncapusulated) Presumptive Tests: a. All are catalase (+) and reduce NO3 → NO2 b. All are oxidase (-) except PLESIOMONAS Plesiomonas is related to Proteus and differentiatied by Oxidase Test (Plesiomonas: Oxidase (+) while Proteus: Oxidase ( -)) Selective: Mac, EMB, DCA, SSA, BSA Ag: capsule (K) – heat labile (destroyed) somatic/cell wall (O) – heat stable; most common in serologic typing flagella (H) – heat labile (destroyed) Vi Ag – removed by Boiling or Heating (for S. typhi)
Lactose Fermenters E. coli Klebsiella Arizona Citrobacter Enterobacter Hafnia *Pathogenic in Stool
Non Lactose Fermenters P-M-P Serratia Salmonella* Shigella* Yersinia* Plesiomonas* – oxidase (+)
Growth on MacConkey: Rapid Lactose Fermenters = Pink Colonies within 24 hours Late Lactose Fermenters = Pink Colonies within 48 hours 1.
Escherichia coli (Colon Bacillus) (LF) Diseases: #1 cause of UTI, #1 Bacteriuria(Cystitis) #1 Gram (-) sepsis #2 neonatal meningitis (K1 antigen) Nosocomial, wound, bacteremia, pneumonia Specimen: Blood Toxin: Endotoxin (systemic) Note: Normally seen in stool! Pathogenic in sto ol = diarrhea. Biochemical Reaction: TSI: A/A GAS(+) IMVIC: + + – – (Indole +) LOA: + + – Greenish metallic sheen on EMB
E. coli vs. E.coli O157:H7 a. MUG E. coli = MUG (+) o E. coli O157:H7 = MUG (-) o b. MacConkey with Sorbitol E. coli = (+) o E. coli O157:H7 = (-) COLORLESS (Does not ferment sorbitol) o E. coli Infections – Biotypes Note: Biotype – based on serological tests; Serotype – based on serological tests Enterotoxigenic E. coli (ETEC)
Traveller’s diarrhea; Watery diarrhea;
Enteropathogenic E. coli (EPEC)
Infantile diarrhea among children (pathogenecity island) Dysentery (shigella) like diarrhea (invasin); Bloody with Mucus stool
Enteroinvasive E. coli (EIEC)
E. coli O6;O8;O25 Cholera-like toxin or heat labile enterotoxin Increases CAMP activity = loss of Na, K and H2O causing dehydration, causing the watery diarrhea E. coli O111, O114
E. coli O124, 143, 164
Sereny test - virulence test for EIEC. (+) Keratoconjuctivitis in mice Hemolytic uremic Enterohemorrhagic E. coli E. coli O157:H7 syndrome (HUS), (EHEC) - Burger is the source of Or hemorrhagic colitis; this strain Bloody urine and = MUG and Mac w/ Verotoxin E. coli (VTEC) diarrhea. Sorbitol (-) Shigella like toxin – verotoxin Acute & chronic diarrhea (aggregative adhesion Enteroaggregative E. coli fimbriae) (EAEC) st Note: For identification of E. coli O157:H7, once found colorless on Mac w/ Sorbitol (1 ), nd perform biochemical test (2 ), then serological test rd (Slide Agglutination: Bacteria + Anti-sera = (+) Agglutination) (3 ).
2.
Enterobacter (LF) TSI: A/A GAS(+) IMVIC: – – + + Urease (-) except E. gergoviae UTI, wound, septicaemia LOA Reaction of Enterobacter E. aerogenes E. gergoviae Hafnia alvei E. cloacae E. sakazakii E./ Pantoea agglomerans
Lysine (+) + + ( –) – –
Ornithine + + + + + –
Arginine ( –) – – (+) + –
3.
E. aerogenes and E. gergoviae is differentiated by Urease: E. aerogenes = Urease (-) o E. gergoviae = Urease (+) o E. aerogenes commonly used as (+) Control for Lysine Decarboxylase E. cloacae commonly used as (-) Control for Lysine Decarboxylase and (+) Control for Arginine Dehydrolase E. aerogenes and E. cloacae can also be differentiated by Arginine test. E. sakazakii and agglomerans are Yellow Pigment Producers
Klebsiella pneumoniae (LF) TSI: A/A (+)GAS; LIA: K/K (LDC +) LOA: + – – IMVIC: – – + + Differentiated with Enterobacter by its: Mucoid Colony; Entero - Dry o Non Motility; Entero - Motile o String Test (+) Urease (+) Malonate (+); = SUM (+) All Klebsiella are Indole (-) except K. oxytoca – Indole (+) (Mistaken as E. coli; can be differentiated by its Mucoid Colony.
Lysine decarboxylase + + + -
5.
Citrobacter (Citrate) (LF) TSI: A/A GAS(+) H2S ( +/- ), LIA: K/A (LDC – ), ONPG: (+) Related to Salmonella UREASE + –
LDC – +
Species: i. C. freundii UTI, pneumonia, endocarditis can cross-react(Ag sharing) with Salmonella typhi
C. freundii S. typhi
ii.
UREASE + –
Mac LF NLF
C. diversus (C. koseri) INDOLE (+) (Like E. coli) nursery outbreak neonatal meningitis mistaken as E. coli
C. freundii E. coli
INDOLE + +
LDC – +
Citrobacter Spp. Differentiation:
Disease: Pneumonia, wound, meningitis, UTI
K. pneumoniae K. oxytoca K. ozaenae malonate (-) K. rhinoscleromatis malonate (+)
Arizona spp. (LLF) – related to Salmonella (Salmonella Arizona) Salmonella that is a Lactose Fermenter (Since Salmonella is a NLF) TSI: A/A GAS(+) H2S (+), ONPG (+), LIA: K/K (LDC +)
Citrobacter Salmonella
Notes:
4.
VP & Urease + + -
Indole + -
C. freundii C. diversus/koseri C. amalonaticus
TSI A/A, GAS (+) H2S A/A, GAS (+) A/A, GAS (+)
Indole – + +
Malonate + + –
6.
Proteus-Morganella-Providencia Group – NLF ALL are PAD (+) ALL are Urease (+) except Prov. alcalifaciens ALL are Indole (+) (like E. coli and C. diversus) except P. mirabilis ALL are LOA ( – – –) except: Morganella and P. mirabilis (ornithine +) Lysine deamination (+) = LIA: R/A (First mentioned to be R/A) ALL can cause WOUND INFECTIION
Proteus – Morganella- Providencia TSI: K/A, GAS( +), H2S (+)
TSI: K/A, GAS( +)
(Swarming) PROTEUS
(NO Swarming) PROVIDENCIA MORGANELLA I CITRATE (+) PROVIDENCIA
Species: P. vulgaris – Indole Positive (Source of OX2 and OX19 – used in Weill Felix Test for Rickettsial Antibody Detection) P. mirabilis – INDOLE NEGATIVE!! (All PMP group are INDOLE (+)!!! Huhu. (Source of OXK) Common Isolate than P. vulgaris Providencia Citrate (+), PAD(+)IMVIC = + + – (+) P. alcalifaciens – UREASE NEGATIVE!! (All PMP group are UREASE (+)!!! Huhu. Morganella –), (Like E. coli) PAD (+), IMVIC: + + – ( Ornithine (+) Has similiarity with the swarmer Proteus (P. vulgaris) = (Morganella and P. vulgaris = the only Ornithine (+) in PMP Group) Morganella vs. Citrobacter! FIGHT!! CITRATE Mac Morganella – NLF Citrobacter + LF
(-) MORGANELLA
Proteus SWARM on BAP , (Burnt Chocolate Agar) GAS(+) H2S (+); LIA: R/A; PAD (+) TSI: K/A Related to PLESIOMONAS! Differentiated by Oxidase Test! Plesiomonas is the only Oxidase (+) of all the Enterobacteriaceae spp.)
Proteus Plesiomonas
M. morgani Prov. Stuartii Prov. Rettgeri Prov. Alcalifaciens P. vulgaris P. mirabilis
OXIDASE – + 7.
#2 UTI, renal stone (urinary calculi) = due to UREASE = virulent factor that can lead to alkaline product Ammonium Carbonate leading to renal st one Considered as CONTIMINANT (If present in urine, perform colony count. If no growth = CONTAMINANT, if there is growth = UTI. Proteus vs. Salmonella! FIGHT!! UREASE KCN Proteus + + Salmonella – –
K/A K/A K/A K/A K/A K/A
TSI Gas(+) Gas(+) Gas(+) Gas(+) Gas(+) H2S Gas(+) H2S
Urease + +/ – + – + +
Salmonella (NLF) MacConkey = Colorless Producing Black Colony in the ff: SSA XLD HEA BSA TSI: K/A GAS(+) H2S (+)(Like Proteus) LIA: K/K REMATCH: Proteus vs. Salmonella! FIGHT!! UREASE KCN Proteus + +
Ornithine + – – – – +
All are GAS PRODUCER (Aerogenic) except S. typhi , S. gallinarum (TSI: K/A H2S(+) All are MOTILE except S. gallinarum, S. pullorum All are H2S(+) and LDC (+) except S. parathyphi A All are LOA (+ + +)
Salmonella spp S. paratyphi A
K/A
GAS(+) H2S (+)
K/A
GAS(+)
S. typhi and S. gallinarium
K/A
GAS(-) H2S (+)
SHIGELLA SPP. S. dysenteriae S. flexneri S. boydii S. sonnei
LDC (+)
H2S (-) LDC (-) LDC (+)
Motility H2S LOA LIA Indole
S. typhi Serology Antigens of S. typhi: Vi, O, H Ag (Requires serologic typing) Without serologic typing, reported as “ Salmonella spp.” Heating removes Vi Ag! Somatic (O) Group where S. typhi is (+) = Group D S. paratyphi A = (H2S and LDC -) Related to Citrobacter. Take note that Salmonella is generally LDC +
Citrobacter Salmonella
UREASE + –
LDC – +
Species and Diseases: S. typhi (MARY = spread the typhoid fever!!!!) o Typhoid Fever st Specimen: 1 week = Blood nd 2 week = Stool (Carrier Sample) Meningitis, Osteomyelitis o S. paratyphi A and B – paratyphoid fever S. paratyphi C (S. cholera suis) – septicimia S. enteritidis – #1 gastroenteritis in Philippines (poultry) Other spp – food poisoning
Invasive Blood culture Related
Shigella spp (NLF) Biochemically inert/Negative (Almost no reaction – Walang kakwenta kwentang bacteria! o Non motile, o Colorless on SSA (Since it cannot produce H2S) o Acetate (-) TSI: K/A only. LIA: K/A (LDC –) Note: Citrobacter, S. paratyphi A, Shigella are ALL LDC ( –) ALL are LOA ( ) except S. sonnei (ornithine +) (Like Morganella and P.
O Ag A B C D
Mannitol Ornithine ONPG + + + + Salmonella Shigella + – + – +++ – – – K/K LDC(+) K/A LDC (-) + – (causing typhoid fever) + – + – Citrobacter E. coli (due to Indole +)
Note: S. dysenteriae is also known as Shiga Bacillus S. sonnei is the only Ornithine (+) among all Shigella spp. S. sonnei cross reacts with Plesiomonas shigelloides. OXIDASE S. sonnei – P. shigelloides +
9.
Serratia marcescens (NLF)
8.
Related to E. coli but acetate (+) Dysentery Dx: culture – fresh stool with mucus flecks and rectal swab of ulcer
Normal in the stool but still need to do Susceptibility Test (since it is an Opportunistic Infection) Ability to produce RED PIGMENT (prodigiosin) at ROOM TEMP (22 or 25’C) at MUELLER HINTON AGAR or NUTRIENT AGAR. ENZYMES: o Dnase: (2 (+) Controls for DNAse Test – S. aureaus & S. marcescens) o Lipase o Gelatinase Opportunistic and Drug resistant (beta lactamase producer) Nosocomial (UTI, bacteremia, pneumo) TSI: A/A or K/A GAS (+/-)(Can be LACTOSE or NON-LACTOSE) NaSIRAan na SIYA, hindi na niya alam kung ano siya. Serratia! Haha weh.
11. Yersinia (NLF) Differential Test for Salmonella, Shigella, and Serratia A.
Y. pestis (plague bacillus) = Coccobacilli! “Safety pin ”, Bipolar Bodies - Wayson Stain is used (Similar to Pasteurella) Antigens: V and W Ag STALACTITE (+): When Yersinia grown in a broth, it grows like a Stalactite UREASE ( –) (Like Entrobacter, P. alcalifaciens, Salmonella) LOA ( – – –) (Like P. vulgaris, Providencia, Shigella) The only Entero spp. that is acquired through insect bite ( Rat flea bite); Bioterrorism Agent Disease: Bubonic/Pneumonic/Septicemic plague; Black death
B.
Y. enterocolitica O O Motile at 22 C but not at 35 C Blood Bank Contaminant TSI: A/A, Ornithine (+), Urease (+), ONPG (+) O Cold enrichment at 4 C (Similar to Listeria that grows at ref temp) Acquired by drinking unpasteurized milk Disease : Appendicitis, Enterocolitis “Bull’e eye colony” on CIN (a selective media for Y. enterocolitica and Aeromonas). To differentiate, OXIDASE Test.
TSI K/A, GAS( +), H2S (+)
K/A
SALMONELLA
SHIGELLA SERRATIA I (Non Motille) SHIGELLA
(Motile) SERRATIA (Red Pigment)
10. Edwardsiella tarda (NLF) Pathogenic in stool, can also be contaminant (like Proteus) TSI: K/A GAS(+) H2S (+) (Similar to Salmonella). Differ in INDOLE. Note: Indole (+) (Like: E.coli, C. diversus, PMP, Shigella)
Edwardsiella Salmonella
IMVIC: + + – – (Similar to E.coli)
Edwardsiella E. coli
INDOLE + –
Lysine decarboxylase (+) Diarrhea, wound, bacteremia
Mac NLF LF
Y. enterocolitica Aeromonas
C.
OXIDASE – +
Y. pseudotuberculosis UREASE (+) LOA ( – – –) Disease: Mesenteric lymphadenitis , sepcticemia
Motility Urease Ornithine Sucrose
Y. pestis – – – –
Y. enterocolitica + (22’ C) + + +
Y. pseudotuberculosis + + – –
VIBRIONACEAE – Vibrio, Aeromonas, Plesiomonas
3.
O129 SENSITIVITY TEST (S) = (+) Zone of Inhibition
All are OXIDASE (+) CATALASE (+), INDOLE (+) All ferment glucose; polar flagella
4.
POLYMIXIN B SUSCEPTIBILITY TEST - to detect the Biotype Classical (S) = (+) Zone of Inhibition El Tor (R)
5.
CHOLERA RED TEST – Nitrate (+) and Indole (+) = (+)RED
VIBRIO “Comma Shaped”, motile (monotrichous) “Darting Motility” OXIDASE (+) except V. mitschnikovii HALOPHILIC except V. cholerae, V. mimicus (seen O129 Susceptible Vinegar – used to counteract Vibrio Alkaliphilic, LOA: ++-, Nitrate reduction (+) 1.
2.
V. cholerae NON HALOPHILIC (1-3% NaCl), Indole (+) Rice watery stool (cholera) Transport Medium: CARY-BLAIR STRING TEST (+) (0.5% Na desoxycholate) SUCROSE Fermenter (Yellow on TCBS ) Vibrio cholera O-1: Pandemic Cause of cholera (Global) Biochemical Tests BIOTYPE CLASSICAL EL TOR S R Polymixin susceptibility + Lysis by bacteriophage – + Chicken RBC agglutination – + Hemolysis of sheep RBC – + VP test –
V. parahemolyticus HALOPHILIC (8% NaCl), BETA HEMOLYTIC, Indole (+), LOA: ++ NON SUCROSE fermenter (green TCBS) KANAGAWA (+): Beta Hemolysis on Wagatsuma Agar #1 Gastroenteritis in Japan (seafood)
Disease V. cholerae
V. alginolyticus V. parahemolyticus V. vulnificus
Cholera (Rice watery stool) Gastroenteritis Gastroenteritis
8% NaCl – (Non Halophilic) + +
Sugar Fermented Sucrose
TCBS Yellow
Sucrose Arabinose
Yellow Green
Lactose
Green
Septicimia, wound
+
NaCl reqt
VIBRIO +
AEROMONAS –
PLESIOMONAS –
Motility Oxidase O129 LOA
+ + S + + –
+ + R + – +
+ + S +++
–
+
–
Note: El Tor – common cause of cholera
Serologic Tests Serotype Anti-Ogawa Anti-Inaba
Ogawa + –
Inaba – +
Hikojima + +
Lab Diagnosis of V. cholera O-1 1. DARKFIELD Microscopy “Darting Motility” 2.
CULTURE a. APW (Alkaline peptone water): (6-8 hrs) An enrichment broth needed to be subcultured in TCBS b. TCBS Selective media for Vibrio
Dnase, Beta-BEM TSI Disease
A/A or K/A A/A (+)Gas (+)H2S All cause diarrhea, wound, septicimia
Note:
Aeromonas and Plesiomonas is seen in Fresh water Vibrio and Plesiomonas can be LF or NLF
A/A or K/A
CAMPYLOBACTER curved, seagull wing Curved, S Shape, Seagull Wing Oxidase, catalase (+) Urease (-), Indoxyl Acetate (+) “Darting Motility” (Similar to Vibrio) Differ in: Morphology (S-Shape vs Comma Shape) o Microaerophilic (5% O2 10% CO2 85% N2) o Growth at 42’C; Zoonotic Suturella wadworthiensis – resembles Campylobacter Species C. jejuni #1 cause of Gastroenteritis in US Guillain Barre syndrome (paralysis) Hippurate differentiates C. jejuni from other spp. C. fetus associated with animal abortion
Diagnosis: Urea Breath Test Rapid test for H. pylori 13 13 C Urea If Urease (+) CO2 (Exhaled) (Uses radiation) Scintillation – counter radiation
NON FERMENTATIVE ORGANISMS
Note: Summary of #1 Gastroenteritis Salmonella enteritidis - #1 in Philippines Vibrio parahemotlyticus = #1 in Japan Campylobacter jejuni = #1 in US O
C. jejuni C. coli C. fetus
37 C + + +
O
42 C + + -
Nalidixic S S R
Cephalothin R R S
Hippurate + -
Gram (-) Rod TSI = K/K, Oxidase (+/-), Mac(+/-) Obligate Aerobes (48-72 hours incubated) Grow best at 37’C except: P. fluorescens and P. putida at RT; Requires AST (drug resistant)
Lab Diagnosis for NFO 1. O-F TEST (2 Test Tubes) Important to detect the type of action of carbohydrates (Increase CHO, Decreased Peptone) Media: HUGH AND LEIFSON MEDIUM (Semi-solid) - inoculate first the organism before the mineral oil, since it has an inhibitory effect to the bacteria 1% glucose, 1% agar, low peptone BROMTHYMOL BLUE – indicator (+) YELLOW (acid) (-) GREEN (no acid) OPEN MEDIUM (Oxidative) Yellow + Yellow + Green –
CLOSED MEDIUM (Fermentative) Green – Yellow + Green –
ORGANISMS
Helicobacter pylori Formerly “Campylobacter pylori, but became “Helicobacter” since its is Helical not curved Pylori = “Pylorus” (seen in stomach Causes Peptic Ulcer and Gastritis Urease (+) - differentiate H. pylori from C. jejuni It can also grow in Butzler/Skirrow Media since it is still a member or Campylobacter
2.
Differentiation of Campylobacter & Helicobacter Oxidase, catalase, Urease microaerophile
GROWTH at 42 C = (+) growth at 35 & 42 C Used to differentiate CONTAMINANTS (P. aeruginosa and P. fluorescens) (+) P. aeruginosa (-) P. fluorescens
3.
CETRIMIDE TEST = 35 C for 7 days Media: Cetrimide Agar/Pseudosel Agar (containing detergent) (+) growth (P. aeruginosa) (-) no growth (E. coli)
C. jejuni
+
-
Media Butzler Skirrow CBAP
OXIDIZER FERMENTER NON UTILIZER (Assacharolytic)
O
NFO Enterobacteriaceae, Vibrio NFO
O
Disease Diarrhea, abortion
O
PSEUDOMONAS SPP
All are OXIDASE (+) and DNASE (-) except S. maltophilia All are MOTILE except B. mallei MacConkey (+) TSI: K/K (No Sugar Fermented since NFO) O-F = Oxidizers (Yellow (O) – Green (F)) Opportunistic infection (Found in environment)
1.
2.
Pseudomonas aeruginosa – B. pyocyaneus Motile = MONOTRICHOUS O-F test = +/- (Oxidise GLUCOSE) “Blue pus Agent” Grape like / Corn Tortillas Odor (Apple Like = Alkaligenes faecalis) Resistant to Disinfectants! NF Organism that has the ability to produce mucoid colony due to its Slime Layer (differ in B. subtilis by its dry colony)
P. aeruginosa P. fluorescens
GROWTH AT 42’C + –
Burkholderia cepacia Contaminants in Alcohol! Associated with IV Drug Users (Important cause of I atrogenic infections) Motile = LOPHOTRICHOUS Lysine decarboxylase (+) (P.aeruginosa is LDC - ) Colonies: Pink colony on Mac (LACTOSE OXIDER) Yellow Colony on (OFPBL) or PC agar YELLOW PIGMENT producer (similar to P. stutzeri )
B. cepacia P. stutzeri
Biochemical Tests: CETRIMIDE (+), ACETAMIDE (+) Mac (+) NITRATE REDUCTION(+) TSI: K/K, LIA: K/A (Lysine Decarboxylase – ) Pseudosel Agar (Cetrimide Agar) is used for the selective isolation of Pseudomonas aeruginosa. O PYOCYANIN Test and GROWTH at 42 C (used to differ from P. fluorescens – also a blood bag contaminant) PYOCYANIN TEST + –
3.
B. pseudomallei P. stutzeri
Diseases:
COLONY Pink/Yellow Wrinkled
OXIDIZER Lactose Oxidizer Non-Lactose Oxidizer
Burkholderia pseudomallei Motile = LOPHOTRICHOUS Melioidosis or Glanders like “Vietnamese Time Bomb” Lactose Oxidizer Wrinkled colony (ashdown medium) (Similar to P. stutzeri)
#1 Opportunistic Infection in Immunocompromised Patients #1 Nonfermentative Organism #1 Cystic fibrosis (Punong puno ng plema. “Siya ay plema na naging tao!” – Aldave) #2 burn (#1 S. aureus)
PIGMENT Yellow Yellow
Diseases: #2 Cystic fibrosis septicaemia, pneumonia Earthy or Dirt Like Odor (drug R)
Pigments: a. PYOCYANIN (Blue-Green Color) – best characteristic to identify P. aeruginosa and differentiate it from other Pseudomonas spp. b. FLUORESCEIN (Yellow-Green Color) (also pigments of P. putida, P. fluorescens; c. Pyorubin, Pyomelanin, Pyoverdin
Wound – Ecthyma gangrenosum (Black) Swimmer’s Ear – “Otitis Externa” Contact lens infection; Sore Eyes Dermatitis – “Jacuzzi”
4.
COLONY Wrinkled Wrinkled
OXIDIZER Lactose Oxidizer Non-Lactose Oxidizer
O
Growth at 42 C (like P. aeruiginosa) O-F = +/- (Lactose Oxidizer)
Burkholderia mallei Only NON MOTILE Pseudomonas Glander’s disease (horses) O-F = +/- (Glucose, Maltose, Lactose)
5.
Pseudomonas stutzeri Brown (buff colored) Wrinkled Colony 6.5% NaCl (+), NO2-N2 gas, Non-Lactose Oxidizer
6.
Stenotrophomonas maltophilia The only OXIDASE (-) and DNAse (+) Lysine Decarboxylase (+) (like B. cepacia) Associated with Catheter Infection (Iatrogenic Infection) Nosocomial Infection O-F = +/ (Glucose, Maltose) Ammoniacal Odor Lavender Green colony
7.
Alkaligenes faecalis Oxidase & catalase (+) Motile (PERITRICHOUS FLAGELLA) Asaccharolytic = O/F test -/ APPLE like “fruity” odor UTI, wound, diarrhea
3.
Moraxella lacunata – Morax Axenfield Blepharoconjunctivitis (Conjuctivitis Agent) Oxidase (+) & catalase (+) Asaccharolytic ; MacConkey (-)(All -ella are negative in Mac, except Salmonella) Mistaken as Neisseria (gram – CB)
Shewanella putrefaciens TSI: K/K H2S; Oxidase (+) The only H2S Producer Pseudomonas
Summary of Sugar Oxidized Pseudomonas Spp. Pseudomonas aeruginosa Burkholderia cepacia Burkholderia pseudomallei Burkholderia mallei Pseudomonas stutzeri Stenotrophomonas maltophilia
M. lacunata Neisseria SUGAR OXIDIZED
Acinetobacter #2 Isolate of NFO next to Pseudomonas Mistaken as Neisseria (gram – CB) OXIDASE Acinetobacter – Neisseria +
OXIDASE (-), CATALASE (+), Non Motile OXIDASE CATALASE – + Acinetobacter – + Enterobacteriaceae
MOTILITY Non Motile Motile
Growth on MacConkey a. A. anitratus (oxidizer) aka: Herella vaginocola b. A. iwoffi (non oxidizer) aka: Mima polymorpha
CHO FERMENTATION – +
4.
Chryseobacterium (Flavobacterium) meningosepticum (+) Oxidase, (+) Dnase, (+) Gelatin Hydrolysos, (+) indole (Generally positive in biochemical tests) Yellow Pigment Producer (FLAVIN) (Chrys = “ Kris Aquino – Yellow” (like B. cepacia and P. stutzeri) Non motile; MacConkey (-) Neonatal meningitis, bacteremia
5.
Eikenella corrodens MacConkey (-) Human bite wound “clenched fist” Corrodes agar, bleach like odor
6.
Kingella spp Cause SBE (HACEK); pits the agar MacConkey (-)
Glucose Lactose Lactose Glucose, Lactose, Maltose Non-Lactose Glucose, Maltose
Other Non-Fermentative Organisms 1.
2.
Acinetobacter Alkaligenes Flavobacterium Moraxella Kingella Eikenella
Oxidase – + + + + +
Catalase + + + + – –
Mac + + –/+ – – –
PARVOBACTERIA
A.
Gram (-) coccobacillus, fastidious (hard to culture) NO GROWTH IN MacCONKEY!
1.
Haemophilus Oxidase (+/-) Medium: CAP (Horse Blood) + 5% CO2 Requires X (hemin) and V (NAD) Satellitism (H. influenzae) Note: NOT cause of influenza (It’s the flu virus) Gram Stain: Gram Negative Cococcobacilli
Differential test for Haemophilus Porphyrin – H. influenzae – H. aegypticus – H. haemolyticus + H. parainfluenzae + H. parahemolyticus + H. paraphrophilus – H. ducreyi + H. aphrophilus
X Factor + + + – – – + –
V Factor + + + + + + – –
The hemolysis of H. haemolyticus and parahemolyticus is seen in Horse Blood Agar. Growth Factor Tests: X and V Test, Porphyrin Test, Sattelitism X AND V TEST – presumptive test for Haemophilus o the media shouldn’t have X and V Factor (Ex. MHA) The X and V factor should be external source (not yet incorporated on the agar) PORPHYRIN TEST (X Factor) - Delta Aminolevulinic Acid (ALA) is o converted to Protoporphyrin (Porphyrin) = (+) Red Fluorescence. Those who are (+) in Porphyrin does not require X Factor.
Example: (CSF should prioritize CAP as its Medium for most probably causative agents are Neisseria or Hemophilus) 1. If CAP (+) BAP (-) Mac (-) = (H. influnzae) 2. Perform X and V Factor Test in MHA st 1. Put the bacteria (1 ) nd 2. Apply the X, V, and X & V Factors (2 ) If the bacteria grows near X, means that bacteria requires X factor for growth, but could also grow in X & V. If the bacteria grow in V, bacteria requires V factor, but could also grow in X & V. For the case of H. influenza, H. ae gypticus, and H.
H. influenzae (Pfeiffer’s bacillus) – GNCB HiB – Haemophilus influenza TYPE B (Serologic Type) Formation of SATELITE around S. aureus in BAP media Used as a POSITIVE CONTROL for BETA LACTAMASE Test Disease: #1 Acute Epiglottis #3 Meningitis (CSF), (#1 Strep. Pneumo, #2 N. meningitides) Pneumonia (among children) (Sputum) Otitis media, cystic fibrosis, conjunctivitis, pneumonia, URT infection RARE isolate of wound infection Lab Diagnosis: 1. CULTURE: CAP, Levinthal Agar, Fildes – “Dew Drop Colony”, Mousy Odor 2. X AND V TEST in MHA 3. SATELLITISM (+) in BAP with S. aureus BAP (contains X Factor) S. aureus (supplies the V Factor) Sources of V factors: 1. S. aureus 2. Strep pneumo 3. Neisseria 4. Candida albicans 4. Porphyrin test (-) (Since it requires X factor)
Note:
H. aprophilus is the only Haemophilus that can grow anywhere (X Factor, V Factor, and X&V Factor)
2.
H. ducreyi (chancroid) – X factor Soft chancre; (hard Chancer = Syphilis) “School of red fish” = cluster of gram negative rods Specimen: Genital sample (non sterile) Growth on CAP + Vancomycin (since the specimen is non sterile) Grows at 33’C Treatment: Erythromycin
3.
H. aegypticus (X and V) Koch’s week bacillus Pink eye conjunctivitis ; Brazilian purpuric fever
B.
Bordetella pertussis Capsule, obligate aerobe Requires CYSTEINE and METHIONINE “Whooping Cough bacillus” ( Lower Respiratory Infection) Vaccine: DPT (Diptheria Pertussis Tetanus Toxoid) 3 Stages: Catarrhal o Paroxysmal – best time to do collection of specimen o o Convalescence Nasopharyngeal swab (carrier). Since B. pertussis is toxic to cotton, it requires Charcoal for the media to remove toxin. Culture media: (NEVER GROW ON BAP, CAP, MAC) Potato Blood Glycerol Agar (Sheep’s Blood) or Bordet Gengou agar – “Mercury Drop or Pearl Like C olony” – not good since Sheep’s blood is the source Regan Lowe (charcoal horse blood) – best since it has charcoal and horse blood is the source. Charcoal cephalexin blood agar (CCBA) – better than PBGA Jones Kendrich (charcoal, yeast extract) Stainer and Scholte, Casamino broth
Spp B. pertussis B. parapertussis B. bronchiseptica
Motile – – +
Urease – + +
Oxidase + – +
Mac, BAP – + +
Note:
B. pertussis is the only human pathogen. The other two are animal pathogen. B. bronchiseptica is KENNEL COUGH BACILLUS Oxidase and Urease are important biochemical tests to differentiate bordetella spp. Clue: B. pertussis is OPPOSITE of B. parapertussis in Urease, Oxidase and Mac,BAP while B. bronchiseptica is (+) on all tests.
C.
Brucella spp No capsule, obligate aerobe, non motile Zoonotic – Erythritol (found on animal placenta) (Important cause of ANIMAL ABORTION) B. abortus – cattle o B. melitensis – goat and sheep o B. suis – swine o B. canis – dog o Used as Ag for Febrile Agglutination Test Disease: Brucellosis, Endocarditis Undulant Fever, Malta Fever Commonly reported laboratory acquired infection Specimen: Blood Culture Media: Castaneda broth (biphasic media) TSB (aerobic since Brucella is aerobic) W medium (Wiskottsin Medium) CAP Rose Bengal (+), 2-mercaptoethanol aggt B. abortus and B. suis are H2S producers. B. abortus causes Bang’s Disease. B. abortus is the only Brucella that requires CO2 Uses Fuchsin and Thionine Dye Inhibition Test for differentiation (-) No growth = Inhibited (+) With growth = Not Inhibited B. abortus is inhibited by Thionine B. suis and B. canis is inhibited bu Fuchsin B. melitensis is not inhibited by Thionine or Fuchsin
Differential test for Brucella B. abortus (Bang’s) B. melitensis B. suis B. canis
Urease + + + +
CO2 + – – –
Thionine – + + +
Fuchsin + + – –
D.
Francisella tularensis Requires CYSTINE (not Cysteine) Related to Pasteurella (Zoonotic) (Non motile, ENCAPSULATED) Obligate aerobe CATALASE (+) BETA LACTAMASE (+) OXIDASE (-) UREASE (-)
HACEK: Agent of SUB BACTERIAL ENDOCARDITIS (SBE), BAP (+) CAP (+), Requires CO2, (+) Mac ( -) All can be seen in Blood Culture = Endocarditis
H
Disease: Tularemia (deerfly, lemming, rabbit, water rat trapper’s disease) – the ONLY PARVOBACTERIA that can be acquired through insect bite (Vector-borne) Lymphadenopathy (Specimen = Lymph Node) Lab acquired infections Culture media: GCBA – GLUCOSE CYSTEIN BLOOD PCA – PEPTONE CYSTEIN AGAR CHA – CYSTEINE HEART AGAR E.
Pasteurella multocida – GNCB “Multocida” = Multiple Killing (Non Motile, ENCAPSULATED like Francisella) Bipolar stain “safety pin” (Similar to Yersinia) MOTL o Yersinia - Insect bite o Pasteurella – Animal Bite (oral flora of certain animals) Oxidase (+) Catalase (+) Glucose (+) Ornithine (+) Indole (+) Urease (+) Grow on BAP but not in MacConkey (-ella) Animal bite wound infection, pneumonia, endocarditis, meningitis, arthritis
C E K
Dew Drop Colony = H. influenzae Mercury Drop (Pearl-Like) Colony = Bordetella Cysteine and Methionine = Bordetella Fuchsin and Thionine = Brucella Cystine = Francisella L-Cysteine = Legionella pneumoniae Sterol = M. pneumoniae
OXIDASE
CATALASE
Does not require X and V
+/ –
–
Actinobacillus Actinomyctemcomitans (Aggregatibacter) Cardiobacterium hominis
Star-like Colony
–
+
Indole (+)
+
–
Eikenella corrodens
Assacharolytic
+
–
Kingella kingae
Twitching Motility
+
–
SPIROCHETES Treponema Leptospira Borrelia
CATALASE – + –
DIAGNOSIS Serology Culture Giemsa serology
DISEASE Syphilis, yaws, bejel, pinta Weil’s Lyme, relapsing fever
Note:
1.
Note:
A
Description Haemophilus aprophilus
All of the above do not grow in culture Leptospira is easily cultured Weil’s Disease (aka Infectious Jaundice) is leptospirosis. Borrelia is Vector BorneLyme Disease is Borreliosis. Giemsa is used as a stain for Borrelia. Giemsa is color Purple.
Treponema pallidum Non cultivable on agar medium Obligate intracellular (rabbit’s testicle) Killed at 4’C after refrigeration Syphilis (Syn – Together) (Pillis – Love) = So this is a product of LOVE ♥♥♥ Primary – hard chancre (can be seen orally) Secondary – condylomata lata (genital lesion), skin rash – BEST time to do serologic test (many Ab present) Latent – asymptomatic (serology) Tertiary – gummas Congenital syphilis – stillbirth, abortion End Result: Cardiovascular Disease and Neurosyphilis (“Kaya pag nagmahal ka,
Torch Test - serologic test for congenital infections Treatment: Penicillin st 1 treatment for Syphilis : Salvarsan (associated Hexheimer Reaction – a post treatment reaction)
3.
Other Treponemes T. pertenue – yaws T. carateum – pinta T. endemicum – bejel
A. Relapsing fever due to Borrelia recurrentis – (EPIDEMIC Relasping Fever) Louse Bite Borrelia anserina, toricatae, parkeri – (ENDEMIC Fever) Tick bite Diagnosis: Presence in Blood or Bone Marrow Stain: Giemsa or Wright Stain
Lab Diagnosis Darkfield microscopy – Corkscrew Motility (CONFIRMATORY) Levaditi Silver impregnation – brown to black Serology a. VDRL, RPR, TRUST (both presumptive and confirmatory) (reagin test) FLOCCULATION b. FTA-ABS (best), TPHA, MHA-TP, HATTS (Trep. Ab test) 2.
Leptospira interrogans icterohemorrhagica Leptospira that is Spiral with hook ends (Like Question Mark – “interrogative”) Cause Weil Disease (coming from animal urine) st 1 week – blood, CSF (acute infection) o nd 2 week – urine (chronic infection) o L. biflexa – non pathogenic leptospira Culture media: Incubate at 30’C for 6 weeks Fletcher’s Medium (contains rabbit’s serum and Fatty acid) Noguchi EMJH (Ellinghaussen McCullough Johnson Harris) Serologic Tests: 1. MAT (Macroscopic Agglutination Test) screening test – – Antigen: KILLED Leptospira + Specimen: Serum (+) Agglutination – 2.
MIT (Microscopic Agglutination Test) Gold Standard – – Antigen: LIVE Leptospira + Specimen: Serum Darkfield Microscope – (+) Agglutination –
Borrelia (Blood spirochete) can be seen on blood smear the spirochete that is arthropod-borne/ acquired through insect bite Spirochete that can be seen in haematology (due to the use of Giemsa Stain) -
B. Lyme disease (B. burgdorferi) #1 Tick Bite Borne Disease in America Vector: Tick bite (Ixodes dammini) O 1 stage – Erythema Chronicum Migrans (ECM) O 2 stage – meningitis O 3 stage – arthritis O Culture on Barber Stoenner Kelly (BSK) at 33 C for 6 weeks
CHLAMYDIA (Former Name: Bedsonia; Now: Chlamydophila)
1.
Obligate INTRACELLULAR (Like Virus), Have both DNA and RNA “ENERGY PARASITE” (Can only live inside the cell) Inclusion body – Diagnostic only (Giemsa Stain) Reticulate body – reproductive that will undergo binary fission o Elementary body - infectious o
Chlamydia trachomatis TRIC agent (Trachoma, Inclusion Conjunctivitis) – leads to blindness LGV (Lympho Granuloma Venerium (#1 STD in US) o Frei Test (Skin Test) = Intracellular – Cell Mediated Immunity NGU (Non Gonococcal Urethritis) Sensitive to sulfonamid Transfer Temperature: 4’C Storage Temperature: -70’C Lab Diagnosis: Glycogen inclusion body ( Halberstadter Prowazeik ) If Glycogen is present = Iodine o If no Glycogen = Giemsa o McCoy (best medium) – a cell media (Gold Standard) DFA (Direct Fluorescent Antibody) – Chlamydia Ag PCR/NAAT – best method
-
2.
Shell Vial Culture System a rapid culture system not only to Chlamydia but also to virus vial that contains coverslip that has monolayers of McCoy and then add the organism. After centrifugation and incubation, transfer the coverslip to the slide and add Iodine stain. a. Brown inclusion Body (Iodine Stain) = Chlamydia b. Fluorescent Stain = Green
Chlamydia psittaci MOT: Inhalation (Bird Droppings) Parrot fever or psittacosis (ornithosis) Man: pneumonia Non glycogen “inclusion body” (Giemsa) – Levinthal Cole Lillie Resistant to sulfonamide Note:
3.
C. trachomatis – Glycogen inclusion body = Halberstadter Prowazeik C. psittaci – Non-Glycogen inclusion body = Levinthal Cole Lillie
Chlamydia “Chlamydophyla” pneumoniae (TWAR – Taiwan Acute Respiratory) Human to human (pneumonia); growth on human lines & Hep-2 cell Immunofluorescence test (DFA), PCR (Best Method)
Rickettsia – Ehrlichia, Orientia, Anaplasma
Obligate intracellular (like Chlamydia) except Coxiella Arthropod borne – commonly TICKS (Chlamydia is sexually, bird and human transmitted) Cross react with Proteus (weil-felix) Site of Multiplication: Endothelial Cell Can also cause DIC Lab Diagnosis Special stain – Gimenez, Macchiavelo Culture – EMBRYONATED EGG is the best culture media Weil-Felix test – Rickettsial Ab Immunofluorescence – more specific test
Ehrlichia
MORULA (Diagnostic of Ehrlichia) Tick transmitted Can destroy leukocytes! Sennetsu fever
Rickettsia spp R. rickettsi R. akari R. typhi
Vector Tick Mite Rat flea
R. prowazekii
Louse
Orientia tsutsugamushi Rochalemia quintana Ehrlichia chaffensis Ehrlichia equi (morulae) Coxiella burnetti
Chigger Louse Tick Tick Tick and Inhalation
Disease RMSF Rickettsialpox Murine typhus (Endemic Typhus Fever) Epidemic typhus, Brill-Zinsser Scrub typhus Trench fever Monocytic E. Granulocytic E. Q fever
Note: Brill-Zinsser disease is a secondary infection Rochalemia quintana ( now Bortanella quinta) – agar (+); trench fever Borrelia vincentii – trench mouth MYCOPLASMA Smallest organism Wall-less (pleomorphic); cannot be gram stained. But if gram stained, they are gram negative coccobacilli or bacilli. Fried egg/Mulberry – if the organism has no cell wall, it has the ability to produce fried egg appearance of colony. M. pneumoniae - Respiratory o M. hominis Genital o U. urealyticum Genital o Requires STEROL (for the rigidity of cell membrane) for growth except acholeplasma. Penicillin resistant because they lack cell wall (not destroyed by penicillin) Size: 10-100 microns (Colonies cannot be seen macroscopically) 1.
M. pneumoniae (Eaton agent) Pleuropneumonia like organism (PPLO) Primary Atypical Agent or WALKING PNEUMONIA (can easily be transferred from one person to another) Fried egg colony (Incubate aerobically w/ CO2) Selective Media: PPLO agar, Edward Hayflick’s Can grow to Chocolate Agar (better) and Blood Agar Confirm: HEMADSORPTION test (attachment of organism to RBC) Serologic test: DFA
BEST: Inhibition of growth by specific antisera M. hominis Large fried egg colony Genital mycoplasma Media: A7/A8, NYCA (like Neisseria) SP4-arginine
2.
String of beads, fluff balls (broth) Fried egg in HEART INFUSION (HI) , SPS sensitive Rat bite fever, Haverhill disease
5.
Spirillum minor/minus Rat bite fever Soduko Fever
6.
Chromobacterium violaceum Violet colored organism, VIOLACEIN; Contaminant NH4 cyanide, Mac – NLF
7.
Cardiobacterium hominis (HACEK organism) Cause SBE, tear drop shape
8.
Capnocytophaga spp Gliding motility (Spreading Colony), Fusiform rod (Like Fusobacterium) Periodontal disease (oral flora) Nitrate (+), Esculin Hydrolysis (+) Catalase (-) and Odixase (-)
Gardnerella /Haemophilus/Corynebacterium vaginalis Oxidase & catalase (-) Not a vaginal flora! (Pathogenic) Cause bacterial vaginosis CLUE CELLS are squamous epithelial cells which can be gram positive or negative (for cytologic exam) Sniff Test: Fishy amine like odor (alk pH – whiff/sniff) (Vaginal Discharge + 10% KOH) then smell. Hmmm AMOY FISHY! LOL o Media: human blood tween 80 agar, V agar, Columbia CAN Treatment: Metronidazole
9.
Bartonella henselae Cat scratch (Cat bite = Pasteurella) Bacillary angiomatosis Peliosis hepatitis
2.
Calymmatobacterium (Klebsiella) granulomatis Safety pin, ENCAPSULATED, NON MOTILE, Non cultivable on agar. Diagnosis: Donovan bodies (Giemsa ) = macrophage with a gram negative rod Disease: Granuloma inguinale (donovanosis)
3.
Actinobacillus actinomycetemcomitans Star like colony (like aggregatibacter) cause SBE Dots and dashes of Morse code
11. Legionella pneumophila Short gram (-) rods, aerobic Catalase (+) Oxidase (+) Motile Require L-cystine and iron for growth Isolated from Air conditioning, water cooling Broadstreet pneumonia & pontiac fever O O Transport = 4 C Storage = -70 C Legionella micdadae – Acid Fast organism
4.
Streptobacillus monoliformis
Disease: Post abortal Past partum fever Pelvic Inflammatory Disease (PID) - Sexually transmitted organisms like Neisseria gonorrhea, Chlamydia trachomatic and M. hominis is asscociated with PID
3.
Ureaplasma urealyticum SMALLEST MYCOPLASMA (Smallest bacteria) T strain (tiny fried egg), Urease (+): (brown); NGU Media: A7/A8, NYCA, SP4-urea Broth: NO HAZINESS since very small despite the growth
Miscellaneous Bacteria 1.
10. Bartonella bacilliformis Destroy RBC, vector: sandfly Carrion’s disease Verruga peruana – skin eruption Oroya fever – anemia
Water Bacteriology Lab Diagnosis: DFA – Legionella Ag Culture: BCYE (Buffered Charcoal Yeast Extract) = BEST MEDIA - blue green o colony/cut glass colony Feeley-Gorman Agar – brown Colony o Stain: Deiterle silver stain = black 12. Listeria monocytogenes Gram (+) rod, Motile at RT not at 35’C, tumbling motility (broth), umbrella like motility (semi solid medium) B-hemolysis on SBAP Cold enrichment at 4’C (like Yersinia) (+) Media: McBride Virulence: Listerolysin O – O2 labile hemolysin Anton test – ocular virulence test (+) Keratoconjunctivitis CAMP test with S. aureus (+) = Block Hemolysis Disease: Meningitis, sepsis, infantiseptica granulomatous, Food poison (coleslaw, cheese, chicken, unpasteurized milk) Fetal abortion 13. Erysiphilothrix rhusiophatiae Gram (+) rod, non motile, H2S producer Catalase (-), Test tube brush (Pipeline Brush) (Semi-Solid) Erysipiloid (Butcher’s cut or Diamond Cut)
Catalase O Motile (25 C) Hemolysis VP H2S prod. Bile esculin & hippurate Gluconate Utilization Media
2. 3.
Presumptive Test Lactose Broth / Lau ryl Tryptose Broth + Water Incubate at 35’C for 24 Hours = Gas (+); 48 hours (-) Confirmatory Test EMB / Endo Agar + Inoculum (24 Hours Inc) = Colony Compeleted Test Lactos Broth Fermenn tube + Org Incubate at 35’C for 24 -48 hours = Acid + Gas (+)
Multiple Tube Fermentation Gold Standard Test (5 Test Tubes) Reported in MPN (Most Probable Number) Positive: >1.1 MPN/100mL; Negative: <1.1 MPN/100MI Stages of MTFT 1. Presumptive Test – Triple Strength Lactose Tube Broth + H2O = (+) Gas (Durham Tube); (-) No Gas after 48 hours 2. Confirmatory Test for Total Coliforms – Brilliant Green Lactose Broth = (+) Gas 3. Confirmatory Test for Fecal Coliforms = EC Broth at 44’C = (+) Gas
Milk Bacteriology
Miscellaneous Gram (+) Bacilli Listeria monocytogenes + + Beta + + + (blue) McBride, Cold enrichment
1.
Erysiphilothrix rhusiophatiae Alpha + - (green) BAP
PATHOGENS Salmonella V. cholerae B. abortus C. diptheriae M. bovis/tb B.anthracis Coxiella FMDV Cowpox virus S. pyogenes
NORMAL FLORA P. syncyanea – Blue Milk F. synxanthum – Yellow Milk P. aeruginosa – Blue Green Milk S. marcescens – Red Milk S. lactis – Souring of Milk B. subtilis – hay bacteria; proteolytic action on coagulated milk Alcaligenes viscosus – slimy or ropy milk
