GENERAL BACTERIOLOGY & DIAGONOSTIC TECHNIQUES Er. Raghvendra Sachan 10/13/2008
CONTENTS STAINING I
GRAM STAIN.
II
SPECIAL STAINING.
1) MOTILITY DEMONSTRATION. 2) BACTERIOLOGY CULTURE MEDIA. 3) STERILIZATION & DISINFECTION. 4) VENEREAL DISEASE RESEARCH LABORATORY (VDRL)TEST. 5) WIDAL TEST. 6) ELISA TEST. 7) DRUG SUSCEPTIBILITY TEST.
STAINING Staining is a biochemical technique of adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. It is similar to fluorescent tagging. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within individual cells. The chemical substances commonly used to stain bacteria are known as dyes. Dyes are classified as natural or synthetic. Chemically dye is defined as an organic compound containing a benzene ring plus a chromophore & auxochrome group. Biological staining is also used to mark cells in Flow Cell Cytometry, and to flag proteins or nucleic acids in Gel Electrophoresis.
GRAM STAIN A Differential stain was developed by Dr. Hans Christen Gram, a Danish physician, in 1884 that is why Gram staining. The bacteria which retain the primary stain (dark blue or violet) are called gram positive, whereas those that lose their crystal violet and counter stained by safranin (red) are referred to as gram negative.
1)-Primary stain: Para Rosaniline Dye- Crystal violet & Methyl violet (if mixed, called Jensen violet).
•
Crystal violet-ammonium oxalate
•
Crystal violet (90% dye content), 2 g Ethyl alcohol (95%), 20 ml
•
Ammonium oxalate, 0.8 g Distilled water, 80 ml
•
Mix solutions A and B. Store for 24 h. before use. Filter through paper into staining bottle.
2)-Mordant: Lugol’s iodine (aqueous solution of iodine that is gram iodine) •
Gram's iodine Iodine, 1 g Potassium iodide, 2 g Distilled water, 300 ml
3) -Decolourisation: Acetone for 2 to 3second & alcohol for 1 minute. (Acid alcohol or acetone alcohol 95%) 4) -Counter stain: Safranin for 30 seconds & methyl red for gonococci & basic Fuschin for 30 seconds. Counter stain alcohol for staining of lock grand. Safranin O (2.5% solution in 05% ethyl alcohol) 10 ml , Distilled water 100 ml.
SPECIAL STAINING 1) STAINING OF VOLUTIN GRANULESAlbert laybourn method used for this staining.
1-Albert stain: Toluidine blue, Malachite green, is mixed with alcohol & then add glacial Acetic acid. Add distilled water to achieve required concentration. 2-Albert iodine: KI & distilled water is used for it. Bluish black colour appears after washing the Albert stain. Preparation of Smear: (a) Heat fix he sample on slide. (b) Cover he smear with Albert’s Stain. (c) Keep it for 15 minutes. (d) Rinse with H2O. (e) Then dip it in Albert’s Iodine. (f) Keep I undisturbed for 1 minute. (g) Wash and dry. (h) Observe. - Bluish-black appearance.
2) Spore Staining Some bacteria are capable of changing in to dormant structures that are metabolically inactive and do not grow or reproduce. Since these structures are formed inside the cells, hence called Endospores. Ferdinand Cohn, a German botanist, in 1828-98 discovered the existence of endospores in bacteria. These are resistant to heat, radiation, chemicals & other agents those are typically lethal to the organism. Impermeable layers called spore coats surround endospores. Some bacterial species, such as Bacillus and Clostridium, form spores in response to unfavorable environmental conditions. These spores are resistant to high temperatures, numerous chemicals, ionizing radiation, and extreme desiccation, the spore begins within the bacterial cell (Sporangium) as an endospore that occupies a characteristic position within the cell (terminal, sub-terminal, or central). As development continues, the sporangium eventually disintegrates, leaving a free spore. The size and shape of the spore is unique to each bacterial species and is often useful in identification.
Spores are weakly acid fast i.e., he resist decolourisation with 0.2% H2SO4 (while Mycobacterium are strongly acid-fast I.e., resist decolourisation with 20% H2SO4). Methods: 1) Modified ZN staining. 2) Malachite green staining. First method is generally not used in Batch Diagnosis. Malachite green staining: Boil water in a beaker and then place the slide carrying the sample over the beaker. The water vapour droplets condense under the slide, thus providing the sample heat. Now put Malachite green on smear for 1 minute. Wash the slide under water. Now put Carbol fuschin (it give red or pink colour) o safranin for 30 seconds. Wash and dry the slide to observe green spores and the rest area as pink.
3) Capsule stainingCapsule are external to the cell is also synthesized partially in the cell is also synthesized partially in the cytoplasm & is usually composed of polysaccharides but may contain other materials, for example, Bacillus anthracis has a capsule of poly-Dglutamic acid. The capsule of specific pathogens can be displaced effectively by the use of antisera specific for a capsule type, & this can provide a presumptive identification of bacterium as on Pnuemococci, Haemophilus influenzae & Meningococci. The cell walls of many bacterial species are surrounded by a polymeric substance referred to as a capsule (if discrete) or slime later (if amorphous). These capsules perform numerous physiological functions, including conferring resistance to phagocytosis and allowing adhesion to sites with favorable environments. These are sometimes also referred to as virulent structures (but are not always present in all virulent forms). The size of such
capsules varies with species and among strains of a species, and may be affected by the composition of the growth medium. Capsules can be clearly observed in: •
Samples by ‘Simple’ staining.
•
Culture by –
(a) Dry India ink Method: This method gives both False-positive and False-negative results hence not preferred. (b) Wet India ink Method: Drop of sample on slide. Add a drop of India Ink. Emulsify the smear. Securely press the cover slip onto the sample and observe. Precaution: •
If the cover slip is not pressed properly onto the sample, then India Ink may move between the cover slip and the sample, thus obscuring the view.
•
Just the right amount of pressure is to be applied while pressing the cover slip, to prevent the sample from undergoing any damage.
4) Intracellular Lipid Staining: Burdon’s Method: After the complete procedure of smear formation, it is stained with Sudan’s Black and then washed with Xylene for 15 minutes. Counter staining with
Safranin / Carbolfuschin for 30 seconds. Appearance of black spots against pink background confirms intracellular lipid in the sample.
5) Spores and Intracellular Lipid Staining: When both endospores and intracellular lipids are to be checked for and differentiated; commonly needed to be carried out in some bacteria.
6) Stains used in case low bacterial count: When sometimes the sample contains low concentration of viable bacteria as n blood or when it is highly entangled in cellular mucoid components as well as when a bacteria is to be viewed inside a virus , then following two procedures carried out: a) Acredine Orange Stain: It is a fluorescent dye thus requires a U.V. Microscope for study. b) Toluidine / Methylene Blue Stain Method: It is an alternative method to Acredine Orange Method, usually carried out U.V. microscope is not available. In this method Toluidine Blue and Methylene Blue are used for staining of Pneumocystis jirove and Haemophilus influenzae respectively.
7) Stains used according to samples: Romanowsky stain & Leishman stain Romanowsky stain: Romanowsky staining was a prototypical staining technique that was the forerunner of several distinct but similar methods, including Giemsa, Jenner, Wright, and
Leishman stains, which are used to differentiate cells in pathologic specimens. It is also called differential stain, as Methylene blue & Eosin is used for it. Ehrlich had used mixtures of acidic and basic dyes for this purpose in 1879. In 1891 Romanowsky and Malakowsky independently developed a technique using a mixture of Eosin Y and oxidized Methylene Blue that was also useful for this purpose. Because the aqueous dye solutions were unstable, methanol was introduced as a solvent, and Leishman (in 1901) and Wright (in 1902) advocated use of methanol as a fixative prior to staining. Giemsa in 1902 improved this technique by standardizing the dye solutions and adding glycerol to increase solubility and stability. The oxidation of Methylene Blue in aqueous solution using heat and alkali produces a mixture of Azure A, Azure B, Methylene Violet and Methylene Blue. Eosin Y is then added to produce a "neutral" dye. The precipitate is then dissolved in a mixture of methanol and glycerol to form a stock solution: this is diluted with water or an aqueous buffer to form a working solution that is used in the preparation of pathology specimens. Leishman's stain: In addition, Leishman stain is used in microscopy for staining blood smears. It provides excellent stain quality. It is generally used to differentiate and identify leucocytes, malaria parasites, and trypanosomas. It is based on a mixture of Methylene blue and eosin. Procedure: In this we used 0.15 gm of Leishman’s powder in 100ml of methanol & grind the mixture again & filter & then in smear, add undiluted Leishman’s stain for 1 minute & do not heat, fix it &for fixation, methanol acts as fixative. After that dilute the stain with distilled water & add the double amount of distilled water & keep it for 12 minute & dilute until smear become pink. Leishman stain uses a methanol solution of staining dyes. 7-10 drops is applied to the slide with the specimen. After 20 seconds, 10-15 drops of a
buffer solution (pH 6.8) is added and mixed with the stain, and then the specimen is left staying for 20-30 minutes, and then washed off with the buffer solution. Leishman stain is named after its inventor, the Scottish pathologist William Boog Leishman. It is similar to and partially replaceable with Giemsa stain, Jenner's stain, and Wright's stain. Like them, it is a version of Romanowsky stain. Acid-Fast Stain The acid fast stain is a differential stain. It was developed by Paul Ehrlich in 1882 which was later on modified by ziehl neelsen & is being used by the present day microbiologist. Bacteria are classified as acid fast if they retain the primary stain after washing with strong acid & appear red or as non-acid-fast if they lose their colour on washing with acids & counter stained by the Methylene blue. Acid fast staining is useful for the identification of members of Mycobacterium tuberculosis, the cause of tuberculosis. A special stain can be used to identify organisms that are compatible with Mycobacterium bovis, the bacteria that causes bovine TB. This is called an acid-fast stain. The cell wall of bacteria belonging to the Mycobacterium family, contain certain structural elements for which the acid-fast stain is specific. Following acid-fast staining, bacteria that take up this stain, including Mycobacterium bovis, will appear as short red or pink rods when examined under a microscope. The finding of acid-fast staining organisms in tissues is only suggestive of infection with bovine TB as other bacteria may also take up the acid-fast stain. More definitive laboratory testing is required to make a definitive diagnosis of bovine TB infection. Chemicals used in Acid-Fast staining : 1. Carbolfuschin – (primary stain) Turns cells, including Acid-fast bacteria, red. Contains phenols, which will solubilize lipids.
2. Acid Alcohol – (decolorizer) Dissolves cell walls which have few lipids. Won’t remove primary stain from Mycobacterium, but will remove it from other kinds of bacteria. 3. Methylene Blue - (counter stain) Dyes the decolorized cells. The resulting blue cells are non-acid-fast bacteria. Ziehl-Neelsen acid-fast staining procedure: 1. Heat fix cells on glass microscope slide. 2. Flood the slide with Carbolfuschin stain. 3. Heat the slide gently until it steams (5 min). 4. Pour off the Carbolfuschin. 5. Wash slide thoroughly with water. 6. Decolorize with acid-alcohol (5 min). 7. Wash slide thoroughly with water. 8. Flood slide with Methylene blue counter stain for 1 min. 9. Wash with water. 10. Blot excess water and dry in hand over Bunsen flame
MOTILITY DEMONSTRATIONMotility in bacteria is achieved by any of several mechanisms. The most widespread mechanism is flagellar movement, which allows travel in a liquid medium and is mediated by special threadlike organelles extending from the cell surface called Flagella.
FLAGELLA Motile bacteria, particularly eubacteria (true bacteria), possess one or more flagella. Flagella are coiled in rigid spirals that revolve around their points of attachment; however, due to the nature of microscopic preparation, flagella are seen as "flat", wavelike organelles. Further, though flagella are many times the length of the bacterial cell, they are about 100 times narrower than most bacterial cells, averaging 0.01 to 0.05 micrometers (µm); hence, flagella are beyond the resolving power of light microscopes and can be seen directly only with an electron microscope. [1]. Most rods and spirilla are motile by means of flagella; cocci are usually nonmotile. A somewhat modified version of the bacterial flagellum is responsible for the movement of the bacteria known as spirochetes. These organisms possess an axial filament. [2] Consisting of two sets of flagella-like fibrils anchored at the two poles of the cell. Another type of movement observed for bacteria is known as gliding motility [3]. It is the sole method of movement of the cyanobacteria and mycobacterium. These organisms can move slowly over solid surfaces. Motility is important in that an organism can swim toward optimal concentrations of nutrients and away from toxic substances - one finds that the longest runs are in the direction toward the nutrient or away from the toxic substance. This type of purposeful movement is called chemo taxis
[4]Other forms of tactic response include photo taxis (movement toward optimal light concentration or wavelength) and magneto taxis (orientation and movement along lines of magnetic force). . Bacteria show four types of flagellation patterns. A) Monotrichous- having single flagella at one end of the cell. B) Lophotrichous- having many flagella in tufts or cultures at one end. C) Amphitrichous- having flagella at both ends, either singly or in tufts. D) Peritrichous- having flagella all over the surface.
Observation of motility •
By naked eyes:
1. In solid media: agar concentration is 2% for New Zealand agar & 4% for Australian agar generally observed on Petriplate
2. In semi solid media: 0.2 to 0.4%. in semi solid media motility is observed in test tube. •
Under microscope:
By wet mount: the sample is taken on slide as a drop and is covered by square cover slip. Petroleum jelly is applied on the edge of the cover slip to prevent the sample from drying. Movement due to air current is thereby prevented. •
Hanging drop method:
Slide should be used with central concave cavity, applied with petroleum jelly & on cover slip also. The slide is first placed onto the cover slip holding the sample drop. Then it is instantaneously upturned to form the hanging drop. It is better microscopy technique than wet mount. Media used in motility demonstration•
Semi solid media: various media used here are as following:1. MBM- simple Motility Broth Media. 2. SIM- Sulphide Indole Motility media. 3. MIO- Motility Indole Ornithine media. 4. O\F- Oxidation-Fermentation media.
Type of motility 1. Darting motility- spontaneous motility observed in vibrio & campylobacter. 2. Tumbling motility- in Listeria, it is motile at 20-250C & non-motile between 30 to 370C. 3. Corkscrew motility- observed in spirochete. Motility of Flagella can be observed via : 1. Staining.
2. Phase-Contrast microscope. Staining used for motility demonstration1. Leifson method 2. Ryu methodThe dye that is used is Rosaniline, which is dissolved in alcohol. Now Tannic acid is added which act as mordant. Now observed under 100X microscope lens (oil immersion).
BACTERIOLOGY CULTURE Requirement: 1. Specimen 2. media 3. inoculating loop 4. incubator Specimen1): Blood culture:- Culture of blood to detect the presence of micro organisms which cause deep-seated infections like bacterimia or septicemia. Localized infection: when the symptoms are present in a localized region then it is a localized infection. E.g. Osteomalitis, Meningitis. Systemic infection: when the causative microorganisms of localized infections move into blood to be detected there from then such an infection is called a systemic infection. E.g. bacterimia and septicemia. Blood culture is done in broth bottles; two respectively for aerobic & anaerobic. This is done to dilute the inhibitory growth factors against microorganisms thus making culture possible (dilution proportion 1:5 or 1:10). Firstly, we take two bottles of 50ml nutrient broth for adult formulation & 30ml for pediatric formulation. After this, incubate for 24 hours & subculture with agar at 370C. 2) Throat swab-It is done to collect sample from the upper respiratory track. Naso pharyngeal swab is done to particularly detect Purtusis. It is carried out to detect pathogenic group A- β haemolytic streptococci. 3) Sputum- It is obtained from lower respiratory tract. Carried out to detect tracheobronchitis, bronchitis & pneumonia. It is used to detect pus cell on epithelial cell
and
various
other
predominant
microorganisms,
such
as
Mycobacterium
tuberculosis.
CULTURE MEDIA Classification of culture media: a) On the basis of composition: a) Natural- urine, milk, juices & body fluids. b) Synthetic- also known as chemical or defined media because exact composition of components are known. c) Semi synthetic- constituent components
are a mix of chemically
defined compounds of known composition and some natural complex compounds as blood agar. b) On the basis of consistency: a) solid b) semisolid c) liquid c) On the basis of complexity: a) Simple b) complex Simple media: i.
Nutrient broth- Basic requirement for growth of microorganisms.
Three existing types are-
all kinds of
•
Meat infusion- ox heart/beef muscles is mixed with water and
kept for 24 to 48 hours. The liquid which is strained off is used as media. •
Meat digest- made by addition of proteolytic enzymes to meat.
•
Meat extract- commercially available peptone is added ii.
Nutrient agar- to nutrient broth agar is added as a solidifying agent which is a polymer of 3,6-anhydro-L galactose and D-galacto-pyranose
Isolation of microorganisms is not possible in broth & it cannot differentiate two different microorganisms. Agar is of two types- Japanese agar & New Zealand agar. 1. Solid- 1% for Japanese or 2% for New Zealand 2. Semisolid- 0.2% for Japanese or 0.5% for New Zealand 3. Firm- 6% for Japanese or 4% for newziland Firm agar-To check the swarming in proteus (motile in nature) and spreading of clostridium tetani in media. Semisolid- it is used to check motility of bacteria (stabbing). It also used as a stock culture. Stock culture is used to reduce the rate of metabolism thus sustaining a large no. of viable microorganisms. It contains 0.4% agar maintained at 40C. Complex media •
Selective media
•
Enriched media
•
Enrichment media
•
Differential media
I. Enriched media: - enriched media are media that allow growth of fastidious organisms because of presence of specific nutritive additives such as heamin, cysteine. Example- Blood agar, Chocolate agar used in case of Neumococcus, H.influenzae, and Loeffler’s serum media.
II. Enrichment media: - it is always a liquid media that favours the multiplication of a particular species of bacteria by incorporating special substances which selectively favours its growth or inhibits its competitors. Example- Salinite F broth, XLD (Xylene Lactose Deoxycolate) media III. Selective media: -This media contains additives that enhances the growth of the desired organism by inhibiting other organism. E.g. Deoxycolate Citrate agar for salmonella & shigella, Lowenstein Jensen media for Mycobacterium tuberculosis, & MacConkey’s agar. IV. Differential /indicator media: - when a culture media containing certain substances helps to distinguish differing properties of different bacteria, it is called differential media, e.g. MacConkey’s agar media. When certain indicator (neutral red or bromothimol blue) or reducing substance is incorporated in media, it is called indicator media e.g. MacConkey’s agar media. Transport media: - It just maintains the microorganisms neither enhancing nor inhibiting the growth. Example- Stuart’s media & Amies media for gonococci, meningococci. Pyke’s media for Streptococcus pyogenes & Borditella purtusis. Thioglycolate broth is used for anaerobic bacteria. Anaerobic media: - It is used for anaerobic bacteria. E.g., Robertson’s cooked meat media (RCM). Meat particles used as a reducing agent & glove box maintain the anaerobic condition, glucose phosphate broth is used as sugar fermentative media. Mycobacterial culture media: Media for mycobacterium: 1. Serum based- e.g., Loeffler’s serum media. 2. Egg based- e.g., Lowenstein Jensen media (LJ) & Dorset media. 3. Agar based media- e.g., Middlebrook media (7H10, 7H11)
4. Liquid middle based media – e.g., Middlebrook media (7H9, 7H12) 5. Blood based media-e.g.,Tarshis media 6. Potato based media- e.g., pawlowsky media
STERILIZATION & DISINFECTIONSterilization and Disinfection in the Laboratory: It is important to distinguish between sterilization and disinfection. Whereas sterilization results in destruction of all forms of microbial life, disinfection results in destruction of specific pathogenic microorganisms. High-level disinfection will kill most vegetative microorganisms but will not kill the more resistant bacterial spores. Commonly used disinfectants such as alcohol, iodophors, quaternary ammonium and phenolic compounds are not effective sterilants and, therefore, are not acceptable for use on items intended to be used in survival surgical procedures. The preferred methods of sterilization are high-pressure steam/temperature (in autoclaves) for items that can withstand high temperature, and ethylene oxide gas for items that cannot withstand high temperature. However, cold chemical sterilants may be used effectively for many items. Physical agentsa) Sun light b) Drying c) Heat d) Filtration e) Radiation f) Sonic & ultrasonic vibration Heat sterilization can be characteristically divided into - dry heat & moist heat. Characteristic Factors of heat sterilization:
a) Nature of heat b) Temperature & heat c) No. of microorganisms d) Type of container Dry heat (mode of action) •
Denaturation of protein
•
Oxidative damage to cells
•
Toxic effect due to elevated levels of electrolyte
Moist heat (mode of action) •
Denaturation and Coagulation of protein
Filtration: - it is used for heat labile solution sterilization example-antibiotic, serum carbohydrate solution. Types of filter1.
Candle filter: it can be cleaned by hypo chloride solution by scrubbing.
E.g., Chamberland filter and Doulton filter 2.
Asbestos filter: It is composed of disposable disc of Asbestos, which is
carcinogenic, so generally avoided. E.g., Seitz Filter and Carbon Filter. 3.
Diatomaceous filter: it can not be scrubbed; layer of algae helps in
filtration; E.g., Barkefield filter & Mandler filter 4.
Sintered filter: Powdery mixture of glass particles of graded size.
5.
Membrane filter: It is composed of cellulose esterase & the average
pore diameter (APD) is 0.15 to 12 µ. Radiation: - it is of two type- ionic & nonionic (UV rays & IR rays). Non-ionic: (a)IR rays- mass sterilization of syringes. (b)UV rays- it is used for closed place, lab, O.T. Ionic:
Is used for cold sterilization ray E.g., Cosmic rays, X – rays & γ-rays. Sonic & ultrasonic is used for particular bactericidal range. Chemical agents: 1. Alcohol- skin disinfection (60%-70%) 2. Aldehydes-formaldehydes, gluteraldehyde. 3. Dye- aniline dye. 4. Halogens. 5. Phenols- 5% phenol for mycobacterium tuberculosis. 6. Gases- E.g., Ethylene oxide. 7. Surface active agent- detergent. 8. Metallic salts- heavy metals.
Outline of the properties of heat decontamination methods. Principle/Conditions Advantages Disadvantages Dry Heat Thermal inactivation: NonLess effective destroys by oxidation corrosive
Hot Air Oven
Uses Materials that are
than moist
damaged by, or
Simple
heat; requires
are impenetrable
design and
longer times
to, moist heat
principle
and/or higher
160-180 C for 2-4
penetrates
temperatures Slow diffusion,
anhydrous
hours
water-
penetration.
materials, such as
insoluble
loading,
oils, greases and
materials
packing critical powders
(e.g., grease
to performance
laboratory
and oil)
not suitable for
glassware,
less
reusable
instruments
corrosive to
plastics
closed containers
o
metals and sharp
instruments than steam
Red-heat
oxidation to ashes
Flame
(burning)
rapid
initial contact
inoculating loops,
with flame can
needles
produce a viable aerosol possibility of Incineration
oxidation to ashes
reduces
accidental fire improper use
(burning)
volume of
may lead to
decontamination
1-60 minutes:
waste by up
emission of
of waste items
temperatures may
to 95%
pathogens in
prior to disposal
smoke
in landfill
exceed 10000C
requires transport of infectious waste excess plastic (>20%) content reduces combustibility Moist Heat
Irreversible coagulation
More rapid of and
(microbial) proteins
more
effective than heat
dry
for
Pasteurization
heating to below
can be used
not reliably
milk and dairy
boiling point
on heat
sporicidal
products
(generally 770C) for
sensitive
some heat-
up to 30 minutes
liquids and
sensitive medical
medical
equipment
devices Tyndallization
Heating to 80-100 C
low cost Resistant
(Fractional
for 30 mins on
spores
consuming.
materials such as
Sterilization)
successive days, with
germinate
To reliably
bacteriologic
increasing Periods in
and are
sporicidal.
media, solutions
between.
killed on the
of chemicals,
second and
biological
Maximum
third days. Minimal
Number some:
materials. Small instruments
temperature
equipment
not practical
and equipment.
obtainable is
required.
for everyday
Oiling
Autoclaving.
0
Time
Heat sensitive
approximately 1000C
lab use.
10-30 mins.
To reliably
Steam under
Minimal
sporicidal. loading and
pressure.
time
packing critical sterile glassware,
210C/15 PSI for 15-
required.
to
media and
90 mins (gravity
cost
performance.
instruments.
displacement
dependable
Yielding dirt
Decontamination
autoclave).
sterilants for must first be
of reusable
320C/27 PSI for 4-20
lab use.
removed.
supplies and
minutes (pre-vacuum
Maintenance
equipment.
autoclave).
and quality
contamination of
control
infectious waste.
essential. damages heat-
Penetration of
sensitive items.
VDRL TEST Definition: VDRL (VENEREAL DISEASE RESEARCH LABORATORY) is a screening test for syphilis that measures antibodies called reagins that can be produced by Treponema pallidum, the bacteria which causes syphilis. However, the body does not always produce Reagin specifically in response to the syphilis bacteria, so the test is not always accurate. The test is similar to the newer Reagin Plasma Response (RPR) test. How the test is performed? Blood is drawn from a vein, usually from the inside of the elbow or the back of the hand. The puncture site is cleaned with antiseptic. An elastic band is placed around the upper arm to apply pressure and cause the vein to swell with blood. A needle is inserted into the vein, and the blood is collected in an air-tight vial or a syringe. During the procedure, the band is removed to restore circulation. Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding. In infants or young children: The area is cleansed with antiseptic and punctured with a sharp needle or a lancet. The blood may be collected in a pipette (small glass tube), on a slide, onto a test strip, or into a small container. A bandage may be applied to the puncture site if there is any bleeding. Why the test is performed ? Syphilis is a highly treatable infection. In addition to screening individuals with signs and symptoms of syphilis or other sexually transmitted diseases, syphilis screening is a
routine part of prenatal care during pregnancy. Several states also require screening for syphilis prior to obtaining a marriage license. Normal Values: The value of a negative test depends on the stage of syphilis that is suspected. Screening test is most valuable in secondary and latent syphilis as it will most likely be positive during these stages. During primary and tertiary syphilis this test may be falsely negative and additional testing may be needed prior to ruling out syphilis. What abnormal results mean? A positive test result may mean you have syphilis. If the test is positive, the next step is to confirm the results with an FTA-ABS test, which is a more specific syphilis test. The VRDL test's ability to detect syphilis depends on the stage of the disease. The test's sensitivity to detect syphilis nears 100% during the middle stages; it is less sensitive during the earlier and later stages. The following conditions may cause a false positive test: •
HIV
•
Lyme disease
•
Certain types of pneumonia
•
Malaria
•
Systemic lupus erythematosus
Special considerations This screening test for syphilis is usually performed on blood. If an individual is suspected of having brain involvement with syphilis (neurosyphilis), the VDRL test may be performed on spinal fluid.
WIDAL TEST Widal test is a serological test widely used for diagnosis of enteric fever. However, as for any other serological test, it has its own limitations as a diagnostic tool. Its main use is in carrying out sero-surveys in a community to know the endemicity of any infection. While using Widal test for the diagnosis of enteric fever, several factors need to be considered for interpretation. 1. Endemicity of enteric fever in an area of investigation. This would determine a cut off titre in that population. 2. Administration of antibodies to the patient will affect the result. 3. Immunization with any typhoid vaccine or a previous infection/exposure will affect the test. 4. The stage of the disease at the time of collection of sample. Early in the disease low antibody titres are found. The antibodies start rising after first week of illness and do so until third-fourth week. Thereafter the titers start falling. 5. Infection with any other gram-negative bacteria may give a false positive reaction. To make any logical conclusion in the diagnosis of enteric fever on the basis Widal test, one must submit a paired serum sample. The first sample taken early in the disease and the second sample at least 2 weeks later. Demonstration of a four-fold rise in antibodies in the paired samples may diagnose acute recent infections. Modified Widal test is a commercially available as slide agglutination test in which IgM antibodies can be detected. The presence of IgM is indicative of acute recent infections. This test needs to be evaluated using bacterial isolation as gold standard.
This test is done for Salmonella typhi & Salmonella paratyphi. This salmonella include gram negative & bacilli, which is motile, non-sporing, non-acid fast, noncapsulated & these belongs to family. It’s belongs to family enterobacteriaceae & having peritrichous flagella & the enzymes present are oxidase negative, catalase positive. It reduces from nitrates to nitrites. Salmonella Typhi is a serovar of Salmonella enterica and the cause of the disease typhoid fever. The organism can be transmitted by the faecal-oral route—it is excreted by humans in faeces and may be transmitted by contaminated water, food, or by personto-person contact Salmonella Typhi is somatic antigen (O antigen) D, Vi positive, Flagellar antigen H. Antigen of typhi – •
H – Flagellar antigen.
•
O– Osmotic antigen (cell wall).
•
Vi – Virulence antigen.
•
Paratyphi A & B
Virulence (Vi) antigen covers the O antigen, thus preventing its detection. Hence, when salmonella is boiled for 30 min 800C to 850C, Vi antigen is removed and O antigen is expressed. If O antigen gives 100 titre & H antigen gives 200 titre, it is significant.The results from the first test are not taken as conclusive, and the Test is again performed after a week to check for rising titre, which is regarded significant only when the titre value doubles. Vaccine – TAB vaccine, this vaccine is used for typhi & paratyphi A & B. This is a killed vaccine
ELISA TEST The Enzyme-Linked ImmunoSorbent Assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. It utilizes two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme. This second antibody gives the assay its "enzyme-linked" name, and will cause a chromomeric or fluorogenic substrate to produce a signal. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the Human Immunodeficiency Virus, HIV test or West Nile Virus) and also for detecting the presence of antigen. Types of ELISA: 1. Indirect ELISA 2. Sandwich ELISA 3. Competitive ELISA Methods: Indirect ELISA The steps of the general, "indirect" ELISA for determining serum antibody concentrations are: 1. Apply a sample of known antigen to a surface, often the well of a micro titer plate. The antigen is fixed to the surface to render it immobile. 2. The plate wells or other surface are then coated with serum samples of unknown antibody concentration, usually diluted in another species' serum. The use of nonhuman serum prevents non-specific antibodies in the patient's blood from binding to the antigen. 3. The plate is washed, so that unbound antibody is removed. After this wash, only the antibody-antigen complexes remain attached to the well.
4. The second antibodies are added to the wells, which will bind to any antigenantibody complexes. These second antibodies are coupled to the substratemodifying enzyme. 5. Wash the plate, so that excess unbound antibodies are removed. 6. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorescent signal. 7. View/quantify the result using a spectrophotometer or other optical device. The enzyme acts as an amplifier: even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve which is typically a serial dilution of the target. Sandwich ELISA
A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows: 1. Prepare a surface to which a known quantity of antibody is bound. 2. Apply the antigen-containing sample to the plate. 3. Wash the plate, so that unbound antigen is removed. 4. Apply the enzyme-linked antibodies which are also specific to the antigen. 5. Wash the plate, so that unbound enzyme-linked antibodies are removed. 6. Apply a chemical which is converted by the enzyme into a fluorescent signal. 7. View the result: if it fluoresces, then the sample contained antigen. Competitive ELISA
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen. 2. These bound antibody/antigen complexes are then added to an antigen coated well. 3. The plate is washed, so that unbound antibody is removed. (The more antigens in the sample, the fewer antibodies will be able to bind to the antigen in the well, hence "competition.") 4. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. 5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.
DRUG SUSCEPTIBILITY TEST Laboratory studies on antibiotic action
DISK DIFFUSION TEST In the laboratory, susceptibility is most often measured using a disk diffusion test. Antibiotic solutions of particular concentrations are dried onto filter paper disks. These are then applied to a lawn of the microbe under examination which has previously been inoculated onto an appropriate solid medium. It is of two typesa) Stokes’ Sensitivity Test b) Kirby-Bauer Test. Stokes’ Sensitivity Test: In the Stokes controlled sensitivity test, a control organism is inoculated on part of a plate and the test organism is plated on the remainder. Disks are placed at the interface and the zones of inhibition are compared. The use of a sensitive control shows that the antibiotic is active, so that if the test organism grows up to the disk it may safely be assumed that the test organism is resistant to that drug.
Kirby-Bauer Test: The Kirby-Bauer test for antibiotic susceptibility is a standard that has been used for years. It has been superceded in clinical labs by automated tests. However, the K-B is
still used in some labs, or used with certain bacteria that automation does not work well with. The basics are easy: The bacterium is swabbed on the agar and the antibiotic discs are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. If the organism is killed or inhibited by the concentration of the antibiotic, there will be NO growth in the immediate area around the disc: This is called the zone of inhibition . The zone sizes are looked up on a standardized chart to give a result as sensitive, resistant, or intermediate. Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug. 2. BROTH DILUTION METHOD An alternative measure of susceptibility is to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of a drug. A series of broths are mixed with serially diluted antibiotic solutions and a standard inoculum is applied. After incubation, the MIC is the first broth in which growth of the organism has been inhibited. The more resistant an organism is, then the higher will be the MIC. The MBC is measured by inoculating the broths used for MIC determinations onto drugfree medium. The MBC is the first dilution at which no growth is observed. Cidal drugs have MBC values that are close to the MIC value for particular organisms. With static agents, the MIC is much lower than the MBC.
The MIC/MBC test of a moderately resistant bacteriostatic drug-
The MIC/MBC test of a moderately resistant bactericidal drug-
.
3. CHEQUERED-BOARD TITRATION METHOD: Most antibiotics are given as single agents. There are, however, occasions when two or more drugs are used in combination. Antimicrobial agents may affect each other when used in combination. The effect may simply be additive. In some cases the activity of one drug enhances that of a second drug. This is referred to as synergy. Alternatively, drugs
may interfere with each other - antagonism. Penicillins and bacteriostatic drugs such as tetracyclines are antagonistic, since penicillins require actively growing cells and static drugs prevent cell growth. In contrast, aminoglycosides are synergistic when used in combination with penicillins. This is important when considering antimicrobial therapy, for example for endocarditis. Synergistic combinations are typically used to treat this condition.
The effects of combining antimicrobial agents 4. E-TEST A non-diffusion based technique employs a performed and predefined gradient of an antimicrobial agent immobilized on a plastic strip. The concentration gradient covers a MIC range across 15 two-fold dilutions of the conventional method. When applied to the surface of inoculated agar plate, the gradient is transferred from the strip to the agar and remains stable for a period that cover the wide variation of critical times associated with the growth characteristics of different microorganisms. After over night incubation, an elliptical Zone of Inhibition centered along the axis of strip develops. The MIC value in gram/ml can be read at the point where the ellipse edge intersects the precalibrated E-Test strip, providing a precise MIC
