BACTERIOLOGY Pioneer notes from Ms. A.Aldave and some added notes Biosafety Biosafety Prevents laboratory acquired infection (F.tularensis and C.immitis) Use of Biosafety Cabinet (BSC Class IIA) BSC uses HEPA filter (0.3um pore size) and negative pressure Biosafety – safety of medical technologist Biosecurity – safety of biologicals (microorganisms) Types BSC Class I air velocity: 75 linear feet/min o o 1 HEPA filter: exhaust air thru HEPA filter product (culture) contaminant o process non-pathogens (BSL-1) o BSC Class II air velocity: 75-100 linear feet/min o o 2 HEPA filter: exhaust and recirculated air thru HEPA filter no product contamination o o vertical laminar flow o must for laboratory/hospitals (BSC Class IIA) process bacterial and fungal pathogens (BSL-2 and BSL-3) o
IIA Exhausts air inside the room, Sef-contained, 70% recirculated air IIB Exhausts air outside the building (radioisotopes, chemicals, carcinogen) BSC Class III supply and exhaust air thru HEPA filter o close cabinet – sealed glove ports o o process viral pathogens (BSL-4)
Classification of Biologic agents (Biosafety Level) BSL-1 No risk M.gordonae B.subtilis BSL-2 Moderate risk Y.pestis Treatment B.anthracis Enterics BSL-3 High risk Mycobacteria Treatment Brucella Francisella Molds BSL-4 High risk Virus No treatment
Bioterrorism categories Category A Highly infectious
Category B
Category C
Moderate morbidity Low mortality Emerging pathogens
Smallpox virus Francisella B.anthracis Ricketssia B.pseudomallei Coxiella Yellow fever Dengue fever Hemorrhagic fever Ebola MERSCOV
Chain of infection: Source, Transmission, Host Bacterial Characteristics 1. Prokaryotic Nuclear body: no nuclear membrane, nucleoid region of the cytosol Cell division: binary fission Cell wall: with peptidoglycan except Mycoplasma and Ureaplasma Cytoplasmic membrane: fluid phospholipid bilayer with carbohydrate and sterol Cell organelles: absent Site of energy production: cytoplasmic membrance Site of protein synthesis: free ribosome 2. Has both DNA and RNA 3. 4 morphology – cocci, bacilli, spiral, comma 4. Measured in micrometer (um) – average size: 0.4-2um 5. Biofilms – property of bacteria to attach on solid surface NUCLEOID No nuclear membrane Chromosome – dsDNA for reproduction Plasmid – extrachromosomal DNA that carries the antibiotic resistant genes; transfer DNA CELL WALL Defines the shape of the bacteria Pathogenicity: M protein: Streptococcus pyogenes
Mycolic acid: Mycobacterium spp. Peptidoglycan (murein layer) consists of glycan chains of alternating N-acetyl-d-glucosamine (NAG) and N-acetyl-d-muramic acid (NAM) Mycoplasma and Ureaplasma – lack cell walls, only contains sterol L-forms in media supplemented Gram positive and gram negative cells can lose their cell walls and grow as L-forms in with serum or sugar to prevent osmotic rupture of the cell membrane Gram positive impermeable to alcohol, thick peptidoglycan, teichoic acid, exotoxin Gram negative negative Permeable to alcohol, alcohol, Thin peptidoglycan, peptidoglycan, LPS, Outer membrance, Periplasm, Lipid A, Exotoxin and endotoxin, endotoxin , Somatic (O) Ag – serotyping CYTOPLASMIC MEMBRANE Selectively permeable Site of energy production (ATP site) Osmotic/permeability barrier Osmotic/permeability Regulate transport of nutrients in and out of cell MESOSOMES Point of attachment for chromosome INCLUSIONS Much granules – MTB Babes-Ernst/metachromatic/volutin n granules as food reserve – C.diptheriae Babes-Ernst/metachromatic/voluti Bipolar bodies – Yersinia pestis RIBOSOMES Bacteria: 70s; Fungi: 80s For protein synthesis Viruses are acellular ENDOSPORE Resting cell, highly resistant to dessication, heat and chemical agents dipicolinate/Dipicolinic Acid Composition: Calcium dipicolinate/Dipicolinic Bacterial genera with spores: Bacillus and Clostridium Target of sterilization Non-reproductive CAPSULE Prevents phagocytosis s erotyping by Quellung reaction Antigenic; on the basis of serotyping Neufeld-Quellung capsular Ag o o (+) capsular swelling due to Ag-Ab o Ex. Strep.pneumoniae, N.meningitides, H. influenza o Serotyping: Somatic O Ag – heat stable Vi Ag and K Ag – heat labile Demonstration Animal tissues and fluids o o Media containing milk or serum Colonies often slimy/mucoid Stains: HISS, India ink/Nigrosin PILI Synonymous to fimbriae Common/Ordinary pili – adherence of bacteria to host cell; virulence factor for Neisseria Sex pili – bacterial conjugation, gene transfer FLAGELLA Tumbling – Listeria Atrichous – no flagellum Gliding- Capnocytophaga Monotrichous – flagellum on one pole Darting - Campylobacter Amphitrichous – single flagellum on each pole Cork screw – Spirochetes (Leptospira, Lophotrichous – tuft of flagella at one or both poles Treponema, Borrelia) Peritrichous – flagella all over the organism Periplasmic flagella – endoflagella/axial filaments Twitching - Kingella Motility best seen at 37C Tests for motility – semisolid medium and stains AXIAL FILAMENTS Spirochete with cork screw motility
Bacterial Virulence Factors Pathogenicity – ability of a microbe to produce disease in a susceptible individual Virulence – relative ability of a microorganism to cause disease or the degree of pathogenicity; usually measured by the numbers of microorganisms necessary to cause infection in the host 1. Adherence factors – pili/fimbriae 2. Anti-phagocytic facrors – capsule and self-component of cell wall 3. Enzymes Coagulase – S.aureus Fibrinolysin – spreading and clotting Hyaluronidase/Duran Hyaluronidase/Duran Renal Factor – spreading 4. Toxins: exotoxin and endotoxin
Source Release
Composition Heat Stability Immunologic Effect
Toxicity Lethal dose
Exotoxin/Enterotoxin Gram positive bacteria/Gram bacteria/Gram neg Released by living bacteria Do not require bacterial death for release Peptide and protein Heat labile except Staphylococcus enterotoxin Converted to toxoid Easily neutralized with antitoxin -Kill host cells and help spread bacteria in tissues -Destroy or interfere with specific intracellular activities
High Smaller dose Tetanus, Lock jaw E.coli, S.aureus Most potent: Botulinum toxin
Endotoxin Gram negative bacteria Released when gram-negative bacterial cell is destroyed Lipopolysaccharide portion of cell envelope Heat stable Not converted to toxoid Not easily neutralized -Disruption of clotting, causing clots to form throughout the body (DIC) -Fever -Activation of complement and immune systems -Circulatory changes that lead to hypotension, shock and death Low Higher dose UTI, Typhoid
Bacterial Growth Factors 1. Nutritional Requirement Carbon o Litotroph/Autotroph – inorganic compound as carbon source (Ex. CO2) Heterotroph/Organotroph – organic compound as carbon source (Ex. Glucose, pathogens) pathogens) o Nitrogen Minerals – sulfur and magnesium Salt 2. Oxygen and Carbon dioxide availability Aerobes - 21% O2 and small amount of CO 2 e.g., Bacillus cereus Obligate/strict Obligate/strict aerobe aerobe - Pseudomonas aeruginosa, aeruginosa, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Brucella Anaerobes - Metabolism is a fermentative type, cannot grow in the presence of O 2 Anaerobes Obligate/strict anaerobe anaerobe - Clostridium perfringens, perfringens, Clostridium botulinum, Veilonella, Actinomyces Facultative Facultative anaerobe anaerobe - e.g., Enterobacteriaceae e.g., Enterobacteriaceae group, Staphylococcus aureus etc. aureus etc. Aerotolerant anaerobe – bacteria not killed by Anaerobic (Gas Pak Jar) exposure to oxygen eg. Lactobacillus 5% CO2, 10% H2, 85% N2 Microaerophilic Microaerophilic - Campylobacter jejuni, jejuni, Helicobacter Microaerophilic/Campy Gas (Candle jar) Microaerophilic/Campy pylori 5% O2, 10% CO2, 85% N 2 Capnophilic – 5-10% CO2 Haemophilus influenzae influenzae, Neisseria gonorrhoeae etc. etc. 3. Temperature
Psychrophilic/cryophilic Psychrophilic/cryophilic = 0-2 0 -2 °C, °C, (refrigerator) BB contam at 4° 4 °C BB contam at RT 1. Listeria – coleslaw food poisoning, milkYersinia enterocolitica Staph. epidermidis borne diseases Pseudomonas fluorescens Bacillus cereus 2. Y.enterocolitica – blood bag contamination Serratia liquifacien (presence of bubbles) Mesophilic - 20-40°C, 20-40°C, (pathogenic) Thermophilic - 40-60°C, 40-60°C, (Thermus aquaticus – PCR enzyme, source of Taq polymerase)
4. pH
pH: 6.5-7.5 a. Acidophilic - Lactobacillus, Fungi b. Neutrophilic - pathogenic bacteria (pH7.2-7.4) c. Basophilic - Vibrio (Alkaline Peptone Water) 5. Moisture – humidophilic 6. Salt concentration a. 7.5% NaCl – S.aureus (MSA contains 7.5% NaCl) b. 6.5% NaCl – E.faecalis ( UTI and wound infection, index of fecal contamination of marine water ) c. 0,3,5,8,10% NaCl – Vibrio (bacteria seen on marine water, V.cholerae – contaminated drinking water) Bacterial Growth Curve 1. LAG phase/Adjustment phase increase in cell size NOT in number n umber little or no multiplication adaptation phase 2. LOG phase/exponential phase increase in growth rate (cell division) susceptible to antimicrobial agents organisms grow at maximum rate (exponential rate) 3. Stationary/Plateau Stationary/Plateau phase NO net growth (death = live cells) Growth ceases because nutrients are exhausted or toxic metabolic products have accumulated 4. Death phase/Period of decline viable count decreases Bacterial Gene Transfer 1. Conjugation plasmid mediated sex pili transposon – mobile, jumping genes gram negative bacteria (ESBL positive)
2. Transduction bacteriophage (virus) mediated Tox gene of C.diptheriae 3. Transformation naked DNA Strep. pneumonia (virulent-avirulent) (virulent-avirulent)
Bacterial Metabolism Respiration (Aerobic process) Kreb’s cycle – aerobil process Electron transport chain – aerobic process Glucose CO2 and H2O Oxidation (Aerobic process) Glucose acid non-fermentative organism (Pseudomonas) Fermentation (Anaerobic process) Glycolysis (EMP): Glucose acid/alcohol Ex. E.coli and S.aureus Culture Media
I.
II. III.
According to physical state/consistency: Preparation Preparation of culture media a. Liquid (broth) – rapid culture, without agar Water – deionized or distilled b. Semisolid – 0.5%-1% agar, for motility pH – 7.2-7.4 Salmonella (+), Shigella (-) Sterile – autoclave Listeria (+), Corynebacterium (-) Dissolved – clear and no particles Steps – weigh, dissolve, sterilize, dispense c. Solid – 2-3% agar d. Biphasic – both liquid and solid (Castaneda for Brucella blood culture medium) According to composition: synthetic/chemically-defined, synthetic/chemically-defined, complex/non-synthetic, tissue Dispensing/Distribution: Dispensing/Distributi on: plated or tubed
IV.
Function and use a. Simple/Basal/Supportive/Gene Simple/Basal/Supportive/General ral Isolation/General Purpose Media Non-fastidious bacteria Nutrient agar/broth, trypticase soy agar/broth – general media for g(+) and g(-) BAP – hemolysis study, general bacterial media Sheep BAP – Streptococcus Horse BAP – Haemophilus spp. Human BAP – Gardnerella vaginalis b. Enriched Media Fastidious bacteria CAP o With X (hemin) and V (NAD) o 0% chocolate, heated BAP o Horse blood o Base medium for TMA c. Enrichment Media Enhance growth of organism Increase lag phase of normal flora, decrease lag phase of pathogen Selenite broth and tetrathionate broth: for Salmonella and Shigella Alkaline peptone water: Vibrio d. Selective Media Inhibitory agents: o Antibiotics – eg.CNA, inhibits g(-), growth of g(+) o dyes, bile salt, alcohol (PEA, inhibits gram negative) Inhibitors for gram positive bacteria: crystal violet, bile salts Inhibitors for gram negative bacteria: potassium tellurite, sodium azide TCBS, SSA, TMA, CBAP – enteric media, recommended for non-sterile samples Examples:
Lowenstein Jensen Media Mueller Tellurite Agar PEA (phenyletyl-alcohol) Thayer Martin, Martin, Modified Thayer Martin, Martin, Martin Lewis, New York City Agar Mannitol Salt Agar Thiosulfate Citrate Bile Salt Agar Columbia CAN (Colistin-Nalidixic) Agar Gonoccoci Agar Gentamicin BAP Bacitracin CAP Cystine Blood Glucose Agar Cystine Tellurite Blood Agar Tinsdale with Potassium Tellurite Cystine Trypticase Agar Charcoal Cephalexin Blood Agar Buffered Charcoal Yeast Extract McCoy TSB Potato Blood Glycerol Agar/Bordet Gengou Agar Cysteine Lactose Electrolyte Deficient Bile Esculin Agar Granada Cetrimide Agar Lysogeny broth Leifson agar, SSA, Onoz agar, Hektoen, XLD Nutrient Agar SMAC
MTB C.diphtheriae for gram positive inhibits gram negative N.gonorrhoeae Staphylococcus Vibrio Gram positive bacteria Gram negative cocci Neisseria Streptococcus pneumonia H.influenzae Francisella C.diphteriae Confirmatory for Neisseria B. pertussis (more preferred than Potato blood agar) Legionella C.trachomatis Brucella spp B.pertussis Urinary tract bacteria Inhibits proteus swarm Group D streptococci Group B streptococci (red) P.aeruginosa Shigella and E.coli Salmonella and Shigella Pigment production E.coli 0157:H7 – colorless colony, negative for sorbitol
e.
Differential Media BAP, MAC, EMB, XLD, HEA (Presumptive ID) Enterobacteriaceae: Enterobacteriaceae: Rapid Lactose Fermenters, Late Lactose Fermenters, Non-lactose Fermenters Selective Differential Media for gram negative – EMB, MAC, HEA, SSA f. Medium for susceptibility testing: Mueller-Hinton Agar g. Media for biochemical testing: TSI, LIA h. Types of Culture Pure culture – one organism, ID/AST o To indicate pure colony: same appearance and ID can be made o Streak plate (best), Pour plate, Selective medium, Animal inoculation Mixed culture – more than 2 organism Stock culture – for quality control (ATCC – American Type Culture Collection)
Equipment, Supplies, and Instrumentation 1. Incubator Set at 35-37’C 18-24 hours: aerobic culture 24-48 hours: anaerobic culture Bacteria: 35’C, Virus: 37’C, Fungi: 25-30’C 2. Inoculating needles Nichrome, platinum No longer than 5cm Nichrome – false positive in ox idase because it contains iron 3. Typing sera – serotyping of bacteria 4. Pasteur pipette – transfer liquid 5. Durham tube water bacteriology Gas detector but not for H 2S (black) 6. Cotton swab – carrier state, 2 samples (for culture and gram stain) Nasopharyngeal Nasopharyngeal swab
N.meningitidis, H.influenza, B.pertusis
Nasal swab: S.aureus Toxic to Neisseria: add charcoal on culture media to detoxify Good for viruses 7. Tuberculin syringe: Mantoux – skin test for TB, test for cellular immunity 8. Commercial ID system Rapid: API (Analytical Profile Index) Enterotube Crystal ID Requirement: Decrease substitute, increase inoculum, unique substrate
Collection, transport, processing and staining of specimens Type of Specimen 1. Sterile – without normal flora 2. Non-sterile – use selective medium Rules 1. Collect before antibiotic theraphy 2. Aseptic collection Betadine/iodophor – blood culture Bottle #2 - CSF 3. Acute stage Leptospirosis – 1st to be positive in blood and CSF Typhoid fever – 1sts to be positive in blood and bone marrow 4. Quantify sufficient, prompt delivery, proper sterile containers
Collection 1. Swab (aerobic)s Cotton - toxic to Neisseria, good for virus Calcium alginate – toxic to virus, good for Neisseria 2. Needle aspiration: aerobic and anaerobic 3. Catheterization: aspirate from catheter tube Foley Catheter – urine IV catheter - blood Storage
1-2 hours delay Refrigerated: stool, sputum, urine, swab except genital (because Neisseria is cold sensitive) Room temperature (not ref): CSF, blood, body fluids, genital swab of N.gonorrhea
Transport Medium
Cary Blair: tool pathogens, Vibrio Stuart’s: bacteria and virus Amies: respiratory Transgrow: Neisseria JEMBEC: Neisseria Todd Hewitt: S.agalactiae (vaginal swab) LIM: modified Todd-Hewitt
Swab Dacron and Rayon – good for bacteria and viruses Swab recommended for:
Transport Temperature
Note: Transport Medium
Nutrients to maintain the growth of organism Buffer to maintain pH Small amount of agar to maintain moisture Transport temperature for anaerobes: RT
Virus: Dacron Urogenital specimen: cotton swab with charcoal Swab not for fungi and anaerobes
CSF – transport (RT), storage (35-37’C) Urine preservative: boric acid Urine, stool, swab, viral specimens, sputum, foreign devices: 4’C Serum for serology: -20C Tissue specimen for long term storage: -70C
Clinical Specimen 1. Blood Iodophore (betadine) and opposite arms Collect before height of fever (at 37.5 or 37.8 bacteria are dead) TSB, BHIB with 0.025% SPS, 1% gelatin o (+) cloudy, gas bubbles, hemolysis, pellicle Blood to broth rati = 1:10 – critical o Adult = 1:5 to 1:10 (1:5 more preferred) Children = 1:10 to 1:20 (1:10 (1:1 0 more preferred) o Subculture on BAP, CAP and MAC o TAT: 7 days o 21 days – Brucellosis, Endocarditis, SBE Note: 0.025% SPS (liquid) – anticoagulant, anti-complementary, anti-phagocytic, neutralizes aminoglycosides and bactericidal effect on serum SPS inhibits: G.vaginalis, Neisseria, S,monoliforms, P.anaerobius o o 1% gelatin: counteracts SPS Automation: Uses BACTEC 9120 o TAT: 5 days o Medium: BACTEC broth (1:10 ratio) with SPS and ARD o (+) Fluorescence Subculture on BAP, CAP, and MAC ID and AST on Vitek 2. Urine Random, catheterized, midstream, suprapubic (anaerobic) Centrifuge: collect sediment for culture and GS Quantitative: use BAP and MAC
o
o o o
For suspected infection like UTI (E.coli – gram negative; Enterococcus and S.saprophyticus – gram positive) Colony count in CFU/ml = number of colony x 1000 (if 0.001mL loop) >100,000CFU/ml = ID and AST of UTI <10,000 CFU/ml – ID only
3. CSF
placed on bottle 2, not refrigerated only incubator Routine test: India ink and gram stain Isolation of: Neisseria and Haemophilus Centrifuge: sediment for culture and GS o BAP, CAP (5-10% CO2) o BHIB, MAC (incubator – No CO2) o India ink method (capsule): CSF Latex agglutionation (capsular Ag)
4. Wound
BAP, MAC, THIO, GS Swab collected at edge after NSS – aerobic culture Needle aspiration aspiration - anaerobic culture
5. Stool
do not GS 1-2grams stool on sterile vial Rectal swab on Cary Blair MAC, BAP, CAP, SSA, TCBS, HEA, XLD SELENITE F (SSA), APW (TCBS), CBAP (42C for 48 hours) Presumptive: oxidase test, biochemical test Confirmatory: Serotyping 6. Respiratory (Sputum, NPS) Processing done on BSC Gram stain (>25PMN, <10EC) Gentamicin BAP – S.pneumoniae Bacitracin CAP – H.influenza (5-10% CO2 incubator, MAC 35C incubator) Do gram stain and AFS Bartlett’s Classification Assess the quality of sputum
Enumerate the number of neutrophils and epithelial cells/LPF
0 score or less – saliva (no inflammation)
>1 score – inflammation/infection
7. Throat swab Sore throat Diptheria BAP, MTM, Gram stain 8. Vaginal urethral swab – CAP, MTM, Gram stain 9. TB culture 1 sputum for GS; 2 sputum for AFS GOLD STANDARD: NALC-NaOH
NALC (n-acetyl-L-cystine): digestant/muc d igestant/mucolytic olytic
2.4% NaOH: decontamination Oxalic acid – Pseudomonas contamination (cystic fibrosis) Anti-formin: chlorox Refrigerated centrifuge centrifuge for 15 mins at 3000xg (4C) Lowenstein-Jensen, Middlebrook 7H11, 7H10
Reporting: (-) = 37C for 8 weeks of no growth (+) = 2-3 weeks growth seen BACTEC = radiometric method TAT:
L-J = 8 weeks (-)
Bactec = 2 weeks (-)
GX (Gene Expert) = 2-3hours
Methods of Studying Microorganism I. Living State (Unstained) 1. Wet mount preparation 2. Hanging drop preparation II. Fixed State (Stained) Staining Methods 1. Simple – 1 dye 2. Differential – 2 dyes (GS, AFS) 3. Special – bacterial structures 4. Indirect/Reflief/Negative – capsule a. India ink test/Borris method b. Nigrossin method GRAM STAIN Purpose Reagents Gram (+) Gram (-) Primary V (Crystal violet) Purple Purple Mordant Purple Purple I (Iodine) “basic” Decolorizer-critical Decolorizer-critical A (Acetone-alcohol) or 95% EtOH Purple Colorless Counterstain S (Safranin) Purple Red *Examine microscopicallt under an oil immersion lens at 1000x for phagocytes, bacteria and other cellular material
Gram (+) becomes gram (-)
Over-decolorization
Old
dying
use of acidic iodine as mordant
penicillin
omit iodine
Gram (-) becomes gram (+)
Under-decolorization
thick smear
Gram stain – general rule 1. All cocci are gram (+) except Neisseria, Veilonella, Veilonella, Moraxella 2. All bacilli are gram (-) except Mycobacteria, Corynebacteria, Clostridia, Bacillus, Lactobacillus, Listeria, Listeria, Erysiphilothrix, Nocardia, Actinomyces 3. All spiral organism are reported as gram ( -) 4. Yeasts are gram (+) Gram positive cocci (aerobes) Gram positive cocci (anaerobes) Gram negative cocci (aerobes) Gram negative cocci (anaerobes) Gram positive bacilli (aerobes) Gram positive bacilli (anaerobes) Gram negative bacilli (aerobes) Gram negative bacilli (anaerobes)
Micrococcus, Staphylococcus, Streptococcus Peptococcus, Peptostreptoccocus, Sarcina Branhamella, Neisseria Veilonella Bacillus, Corynebacterium, Erysipelothrix, Listeria, Mycobacterium, Nocardia Actinomyces, Clostridium, Propionobacterium Acinetobacter, Aeromonas, Alcaligenes, Bordetella, Brucella Enterics, Francisella, Legionella, Pasteurella, Pseudomonas, Vibrio Fusobacterium, Bacteroides
NOT Gram stain 1. Chlamydia/Ricketssia – intracellular 2. Mycoplasma/Ureaplasma – no cell wall 3. Spirochete – can’t resolve by bright field
Note: Acridine orange (nucleic acid) BURKE’S MODIFICATION OF GS for metachromatic granules
ACID FAST STANING METHODS Mycolic acid – responsible for acid fast resistance Acid fast organisms
Mycobacteria, Nocardia spp Cryptosporidium – modified AFS (No heating, 1% H2SO4) Others: Rhodococcus, Tsukamurella, Legionella micdadei, Isospora, Sarcocystis, Cyclospora, Gordonia
Purpose
Ziehl-Neelsen (Hot) – CAM Best AFS
Kinyoun (Cold) – (C-A-M) Tissue AFS
Primary Mordant Decolorizer Counterstain Result
Auramine-Rhodamine Auramine-Rhodamine (Fluorochrome) SENSITIVE AFS Auramine-Rhodamine
Carbol-Fuchsin Carbol-Fuchsin Heat Pheno, Tergitol 3% acid alcohol 3% acid alcohol 0.5% acid alcohol Methylene blue Malachite green 0.5% KMNO4 – quenching agent AFO-Red AFO – red AFO – yellow fluorescence NAFO-Blue NAFO – green NAFO – NO fluorescence *Screening at 400 magnification and confirm all suspicious (red) organisms at 1000 magnification Other acid fast stain
Pappenheims (urine) = M.smegmatis (blue), M.tuberculosis (red) Baugmarten’s (tissue) = M.leprae (red), M.tuberculosis M.tuberculosis (blue) Fite Faraco stain = M. leprae (hematoxylin as counterstain)
Special Stain
Metachromatic granules – culture on Loeffler’s, Pai medium and stain (Neisser’s, Albert, Ljubinsky, LAMB) Spore – culture on Bap and stain (Dorner’s, Acetic acid, Wirtz -Conklina Capsule – specimen (Hiss/India Ink) Flagella – culture (Gray Leifson, Fischer-Conn) Nuclei Acid – Fuelgen method Ricketssia – Gimenez, Macchiavelo, Giemsa Bacillus anthracis – M’Fadyean Spirochetes – Levaditis, Fontana-Tribondeux Calcouflour whitet – binds to chitin cell wall (fungi – yellow green fluorescence) Acridine orange - stains nuclei acid acid (fungi – green fluorescence; bacteria-orange/red fluorescence) LPCB – Aman stain for fungal structure
Non-staining methods 1. LANA (L-alanine-4-nitronilide) (+) Yellow color For gram negative
2. String’s test 3% KOH (+) string formation For gram negative Culture the bacteria first
Types of Microscopy Resolution – extent to which detail of the object is maintained (cellular details) Resolving power – closest distance the two objects can be distinguished from each other 1. Bright-Field GS, AFS, KOH, most common microscope Used for stained and unstained samples 2. Dark-Field Motility of Spirochetes Also used for Fluorescent stains Higher resolving power than bright field 3. Phase Contrast Inclusion bodies seen on virus and Chlaymydia, living cells/natural state, Microlymphocytotoxicity Microlymphocytotoxicity test for HLA Good for KOH mount
4. Fluorescent Requires fluorescent stain (Calcofluor white, acridine orange) UVL – provided by mercury arc lamp Substitute: Dark Field microscopy a. Electron Microscopy – viral morphology b. Transmission Electron Microscopy – internal structure c. Scanning Electron Microscopy – external structure Sterilization Sterilization – sporocidal, all organisms are killed sterilization , CJD, agent, Prions – no nuclei acid, most resistant to sterilization, Envelope virus – most sensitive to sterilization, most easily destroyed Killing of MTB in sputum o Boiling - 10 minutes o Direct sunlight – 20-30 hours Dried sputum 0 6-8 months o o 5% phenol – 24 hours Sterilization Sterilization Method by MOIST HEAT 1. Autoclave/Steam Under pressure Autoclave tape o Heat sensitive indication o Control: B.stearothermophilus o Blackline if it reaches 121’C Denatures protein
121’C 15lbs psi for 15 minutes
2.
3. 4.
5.
Fractional Sterilization 1st day = VEGETATIVE CELLS 2nd day = SPORES 3rd day = REMAINING CELLS
Best sterilization and waste disposal Sterilize bacteriologic media (gauze) Sterilize most contaminated microbiological microbiological material Infectious medical waste
121’C at 15psi for 60 minutes 132’C for 30 to 60 minutes Inspissation Sterilize CHON containing medium (L- J, Loeffler’s) 75-80’C for 2 hours on 3 days Tyndallization – 100’C for 30 minutes on 3 days Boiling 100’C for 30 minutes Non-sporocidal (disinfection) Pasteurization for milk Phosphatase - test for success of pasteurization, pasteurization, phosphatase should be negative Grade A milk – 75,000 before pasteurization 15,000 after pasteurization Bacteria seen on unpasteurized milk: Listeria, Brucella, Y.enterocolitica, M.bovis a. Low Temperature Holding (LTH) – 62’C for 30 minutes b. High Temperature Short Time (HTST) – 72’C for 15 seconds
Sterilization Sterilization Method by DRY HEAT 1. Hot Air Oven QC: Bacillus subtilis Dry hot air at 170- 180’C for 2 hours Glass wares, cotton swabs, oils, powders 2. Incineration – not used 3. Cremation - to control disease 4. Flaming – needles, use 70% alcohol prior to flaming 5. Gas Used for machines For heat labile materials: ethylene oxide For heat labile solution: cellulose membrane filtration
Others Cold temperature/freezing temperature/freezing – for preservation, preservation, bacteriostatic Lyophilization/freeze drying – best preservation for bacteria for many years Osmotic pressure – food preservation, use of high concentration of salt and sugar Dessication – food preservation, aka dehydration Ultraviolet Light Acts on DNA (air and water) Reduce airborne infection For hospital room, laboratory, BSC Produces pyrimidine dimer DNA that can lead to cancer 6. Ionizing radiation - disposables 7. Filtration Air and water HEPA filter: BSC 0.3um Cellulose membrane – liquid filter, 0.22um 8. Gamma radiation - sterilization of plastic wares/plates 1. 2. 3. 4. 5.
Disinfection/Antiseptic Disinfection – non-sporocidal, some organisms are killed, kills pathogen Disinfection for living things, Antiseptic for non-living things 1. 10% Sodium hypochlorite (Chorox) Spillage disinfectant 10-30 minutes contact 2. Iodine/iodophor – sporocidal, best antiseptic for blood culture 3. 70% ethyl alcohol – non-sporocidal, cannot destroy viruses 4. 3-6% Hydrogen peroxide – cleansing of wound 5. 1% silver nitrate Crede’s prophylaxis Prevent gonococcal opthalmia neonatorium 6. Dyes – inhibits gram positive, anti-fungal 7. Formaldehyde - reject for bacterial culture because aldehyde is sporocidal 8. Glutaraldehyde (Cidex) cold sterilant for surgical equipment sterilization 9. Phenol (Carbolic Acid) - standard disinfectant 10. Lysol (Cresol) 11. Zephiran – benzalkonium chloride 12. Quats (Quaternary ammonium) Inactivated by organic materials Note:
Standard precautions - Blood and body fluids precautions must be ob served for all patient ’s ’s blood and body fluid specimen Universal precautions – all human blood and all other body fluids that contain visible blood precautions must be observed
Antimicrobial Agents Antimicrobial Antagonistic = 1>2 (single drug) Synergistic = 2>1 (combined drugs) Minimum Bactericidal Concentration or Minumum Lethal Concentration Antibiotics – derived from bacteria or fungi, not used for viruses Eg. Penicillin, Cephalosporin, Streptomycin o Streptomyces – fungus like bacteria, common source of antiobiotics (Ex. Streptomyces, Nystatin) o Chemotherapeutics – chemically synthesized (SXT) Broad spectrum – wide range of bacteria (Tetracyline- for gram pos and gram neg) bacteria (Van – for gram pos only, only , not sensitive for gram neg) Narrow spectrum - limited number of bacteria Bactericidal – Penicillin, Vancomycin, Aminoglycoside, Quinilnes, Metronidazole, TB d rugs (RIZES) – Rifampin, Isoniazid, Pza, Ethambutol, Streptomycin Bacteriostatic – Chloramphenicol, Tetracyclin, Erythromycin, Clindamycin, SXT
Cations effect o Increased Ca and Mg ions – False resistant to aminoglycoside in P.aeruginosa o Increase thymidine or thymin – False resustatn to SXT in Enterococci
A. Cell Wall inhibitors – sensitive for gram positive organisms 1. Penicillin – Penicillum notatum (gram pos) 2. Cephalosphorin – Cephalosporium (Acremonium) 3. Vancomycin – Streptomyces, treatment for MRSA (Penicillin R, Vancomycin S) 4. Aztreonam – Chromobacterium violaceum 5. Imipenem - carbapenems 6. Penicillinase Resistant Pen = Methicllin, Cloxacillin, Nafcillin, Oxacillin B. Cell Membrance Inhibitors 1. Bacitracin 2. Colistin, Polymixin (Bacillus) 3. Amphotericin B, Nystatin (Streptomces), Imidazole, Clotrimazole (Anti -fungal) Structure destroyed by antibiotics: cell membrane (cell wall is not destroyed) C. Ribosomes (CHON) Inhibitors – broad spectrum, for gram pos and gram neg 1. Aminoglycosides – gentamicin, kanamycin, tobramycin, netilmicin, amikacin, streptomycin 2. Tetracycline, chloramphenicol – bone marrow suppression 3. Erythromycin (macrolide) substitute for patients with allergy to penicillin eg.Clindamycin, Lincomycin D. Nucleic Acid (DNA) Inhibitor 1. Mitomycin, Quinolones (levofloxacin, norfloxacin) norfloxacin) 2. Metronidazole anaerobes, anti-protozoa, for amoebiasis G.vaginalis Ex. Flagyl 3. Sulfonamide-Trimethopri Sulfonamide-Trimethoprim m (SXT) – inhibits folic acid (Ex. Rifampin) Antibiotic Susceptibility Susceptibility Testing 1. Micro/Macrobroth Micro/Macrobroth Dilution for anaerobes MIC and MBC Reference method 2. Agar dilution – many organisms vs single drug 3. Disk diffusion one organism vs multiple drugs for aerobes or facultative aerobes 4. Epsilometer test (E-test) antibiotic strip diffusion MIC test filter paper strip with antibiotic MIC – ellipse zone at intersection 5. Automated System – Vitek 6. Disk Elution Test – for Mycobacteria 7. Schlichter Test Serum bactericidal test Measure activity of the antibiotic in the patient’s own serum against an infecting organism Kirby Bauer Disk Diffusion – disk agar diffusion, aerobes/facultative, use of filter paper disk Standard inoculum 1.5 x 108 organisms/mL Medium Mueller-Hinton Agar pH 7.2-7.4 Depth 4mm Condition Aerobic, no CO2 Temperature 35-37’C 35-37’C (MRSA – 35’C) Incubation time 16-18 hours Standard McFarland Std (1% H 2SO4 & 1.175% BaCl2) 0.5 concentration for bacteria 1.0 concentration for fungi Antiobiotic Disk 6mm Bacterial count method Petroff-Hausser
General AST media MRSA S.pneumoniae and N.meningitides Haemophilus Neisseria Mycobacteria Anaerobes Notes to remember:
AST Media Mueller-Hinton Agar MHA + 2% NaCl MHA + 5% Sheep’s Blood Haemophilus Test Medium GC Agar Middlebrook 7H10 Wilkins-Chalgren broth and agar
Disk distance -15mm 150mm – 12 discs; 100mm – 5-6discs Ignore swarming Indirect relationship between organism and zone of inhibition Susceptible: decrease organism, increase zone of inhibition o o Resistant: increase organism, decrease zone of inhibition
False Sensitive Delay of 15 minutes before incubation Increase drying Thin medium
False Resistance Delay of 15 minutes before disc application Incrase moisture Thick medium
AST For Mycobacteria Mycobacteria
MDR-TB ® to INH, Rifampin Rifampin XDR-TB ® to INH, Rifampin, and Quinolone
1. Disc elution 2. Bactec (RIA) and MGIT (IFA) – AST and Rapid culture system 3. Gene Expert – ID and AST Drug Resistance Mechanism 1. Inactivates the antibiotic (beta-lactamase) – destroys penicillin 2. Decrease permeability 3. Acquired resistance – SUPERBUGS: chromosome (MRSA) and plasmid mediated (ESBL) 4. Intrinsic resistance Novobiocin ®: S.saprophyticus SXT and Tetracycline ®: P.aeruginosa 5. Modifies target site 6. Multi-drug resistance (MDR) pump Drug Resistant Test GRAM NEGATIVE BACTERIA Extended-Spectrum Beta Lactamase (ESBL) AmpC Beta Lactamase – g(-) and g(+) Modified Hodge Test
Metallo-Beta Lactamase (MBL) Test
GRAM POSITIVE BACTERIA mecA mediated Oxacillin Resistance Inducible Clindamycin Resistance
Resistant
(+) Result
Penicillin Monobactams Cephalosporin Penicillin Cephalosporin Carbapenems (imipenem)
“Keyhole effect” Elleptical clearing on Cefotazime-Clavulanic Cefotazime-Clavulanic acid-Aztreonam disk Cefoxitin screen Flattened edge zone on Cefotaxime disk Carbapenemase Carbapenemase screening (+) Clover leaf like Swab MHA with E.coli Klebsiella pneumonia – gram neg bacilli Keyhole between imipenem and EDTA Pseudomonas aeruginosa and S.maltophilia – gram negative bacilli
Penicillin Cephalosporin Cephamycin Carbapenems Oxacillin and penicillin Macrolide (Erythromycin) Clindamycin
Oxacillin and Penicillin resistant MHA with NaCl Staph and Strep Flattening (D-zone) of clindamycin zone
