GENERAL BACTERIOLOGY & DIAGONOSTIC TECHNIQUES

Er. Raghvendra Sachan10/13/2008

CONTENTS

1) STAINING -

I GRAM STAIN.

II SPECIAL STAINING.

2) MOTILITY DEMONSTRATION.

3) BACTERIOLOGY CULTURE MEDIA.

4) STERILIZATION & DISINFECTION.

5) VENEREAL DISEASE RESEARCH LABORATORY (VDRL)TEST.

6) WIDAL TEST.

7) ELISA TEST.

8) 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 GRANULES-

Albert 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 staining-

Capsule 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-D-

glutamic 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 DEMONSTRATION-

Motility 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", wave-

like 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

non-motile. 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 demonstration-

1. Leifson method

2. Ryu method-

The 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

Specimen-

1): 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 all kinds of microorganisms.

Three existing types are-

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 & DISINFECTION-

Sterilization 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 agents-

a) 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 filter-

1. 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 Uses

Dry Heat Thermal inactivation:

destroys by oxidation

Non-

corrosive

Simple

design and

principle

Less effective

than moist

heat; requires

longer times

and/or higher

temperatures

Materials that are

damaged by, or

are impenetrable

to, moist heat

Hot Air Oven 160-180oC for 2-4

hours

penetrates

water-

insoluble

materials

(e.g., grease

and oil)

less

corrosive to

metals and

sharp

instruments

Slow diffusion,

penetration.

loading,

packing critical

to performance

not suitable for

reusable

plastics

anhydrous

materials, such as

oils, greases and

powders

laboratory

glassware,

instruments

closed containers

than steam

Red-heat

Flame

oxidation to ashes

(burning)

rapid initial contact

with flame can

produce a

viable aerosol

possibility of

accidental fire

inoculating

loops, needles

Incineration oxidation to ashes

(burning)

1-60 minutes:

temperatures may

exceed 10000C

reduces

volume of

waste by up

to 95%

improper use

may lead to

emission of

pathogens in

smoke

requires

transport of

infectious

waste

excess plastic

(>20%)

content reduces

combustibility

for

decontamination

of waste items

prior to disposal

in landfill

Moist Heat Irreversible

coagulation of

(microbial) proteins

More rapid

and more

effective

than dry

heat

Pasteurization heating to below

boiling point

(generally 770C) for

up to 30 minutes

can be used

on heat

sensitive

liquids and

medical

devices

not reliably

sporicidal

milk and dairy

products

some heat-

sensitive medical

equipment

low cost

Tyndallization

(Fractional

Sterilization)

Heating to 80-1000C

for 30 mins on

successive days, with

increasing Periods in

between.

Resistant

spores

germinate

and are

killed on the

second and

third days.

Time

consuming.

To reliably

sporicidal.

Heat sensitive

materials such as

bacteriologic

media, solutions

of chemicals,

biological

materials.

Oiling Maximum

temperature

obtainable is

approximately 1000C

10-30 mins.

Minimal

equipment

required.

Number some:

not practical

for everyday

lab use.

To reliably

sporicidal.

Small instruments

and equipment.

Autoclaving. Steam under

pressure.

210C/15 PSI for 15-

90 mins (gravity

displacement

autoclave).

320C/27 PSI for 4-20

minutes (pre-vacuum

autoclave).

Minimal

time

required.

cost

dependable

sterilants for

lab use.

loading and

packing critical

to

performance.

Yielding dirt

must first be

removed.

Maintenance

and quality

control

essential.

damages heat-

sensitive items.

Penetration of

sterile glassware,

media and

instruments.

Decontamination

of reusable

supplies and

equipment.

contamination of

infectious waste.

  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 person-to-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 non-

human 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 antigen-

antibody complexes. These second antibodies are coupled to the substrate-

modifying 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

1. 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 types-

a) 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 drug-

free 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