Ebook Practical manual of medical microbiology: Part 2

Part 2 book “Practical manual of medical microbiology” has contents: Bile solubility test, antimicrobial susceptibility testing, brucella agglutination test, treponema pallidum haemagglutination assay, enzyme linked immunosorbent assay, experimental animals, fungal slide culture, medical entomology,… and other contents.

This test detects the presence of enzyme nitrate reductase whichcauses the reduction of nitrate to nitrite in the presence of a suitableelectron donor Almost all Enterobacteriaceae members reduce nitrate.Nitrate reduction can be detected by an appropriate colorimetricreagent

Requirement

1 Medium

Potassium nitrate - 0.2 gm

Distilled water - 1 litre

Tube in 5 ml amounts and autoclave at 121oC for 15 min

2 Test reagent

Solution A - Dissolve 8 gm sulphanilic acid in 1 litre of

acetic acid 5 mol/litSolution B - Dissolve 5.0 gm of α-naphthylamine in litre

of acetic acid 5 mol /litre immediately beforeuse, mix equal volume of solution A and B

to give the test reagent

Nitrate Reduction Test

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Nitrate Reduction Test 119

Method

• Inoculate the medium with test organism

• Incubate at 37 oC for 96 hours

• Add 0.1 ml of the test reagent to the test culture

Result

A red color within a few minutes - Nitrate reduction positive

e.g Esch.coliNo.red color - Nitrate reduction negative

e.g Acinetobacter

List of media used for H 2 S detection

1 Bismuth sulfite agar

2 Triple sugar iron agar (TSI)

3 Lysine iron agar

4 Lead acetate agar

5 Kigler iron agar

6 Deoxycholate citrate agar

Hydrogen Sulphide

Production Test

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Hydrogen Sulphide Production Test 121

Method 2

Filter paper method

• Prepare a Whatman No.1 filter paper impregnated with leadacetate (10% solution)

• Expose the filter paper above culture by placing filter paperbetween the cotton plug and test tube

Result

Blackening of the lead H2 S positive (Citrobacter,

No blackening of the lead H2S negative (e.g Esch coli,

acetate paper Klebsiella)

Some bacteria, particularly those growing naturally in anenvironment exposed to urine, may decompose urea by enzymeurease and liberate ammonia

Urea → Ammonia + CO2 + Water

The production of the enzyme urease is detected by growing theorganisms in the presence of urea and testing for alkali (NH3)production by means of a suitbale pH indicator (Fig 30.1)

No colour change Urease negative (e.g Esch.coli)

Figure 30.1: Urease test (For colour version, see Plate 8)

Some bacteria can utilise simple organic salts like sodium citrate orsodium acetate as the sole source of carbon and energy source forgrowth and an ammonium salt as the sole source of nitrogen Duringsuch utilisation CO2 is liberated which makes the medium alkaline.Koser’s liquid citrate medium or Simmon’s citrate agar may be used

Requirements

1 Medium: Simmon’s citrate agar medium (This is a modification

of Koser’s medium with agar and an indicator added) (Fig 31.1).Sodium chloride 5 gm

Citrate Utilisation Test 125

Adjust the pH to 6.8 Sterilise by autoclaving at 121oC for 15 min.Allow to set as slopes in test tubes

Some bacteria ferment carbohydrates with the production of acetylmethyl carbinol or its reduction product 2, 3 butylene glycol Thisbreakdown product when reacts with Alpha-Naphthol in alcohol

in the presence of alkali gives a cherry red colour

This test is usually done in conjunction with the methyl red testsince the production of acetyl methyl carbinol or butylene glycolusually results in insufficient acid accumulation duringfermentation to give a methyl red positive reaction An organism ofthe Enterobacteriaceae family is usually either MR positive and VPnegative or MR negative and VP Positive (Fig 32.1)

Voges-Proskauer Test (VP Test) 127

Method

• Inoculate the liquid medium

• Incubate at 37% for 48 hours

• Add 1ml of 40% potassium hydroxide and 3 ml of alpha naphthol

• Shake well and keep it in the rack for 10 minutes

Result

Pink or cherry red colour VP Positive (e.g Klebsiella)

Yellow colour VP negative (e.g Escherichia coli)

Figure 32.1: VP test (For colour version, see Plate 9)

This test is used to detect the production of large amount of acidduring the fermentation of glucose and the maintenance of conditionssuch that the pH of an old culture is sustained below a value ofabout 4.5 The acidity can be determined by adding a few drops ofmethyl red solution, as indicator (Fig 33.1)

2 Methyl red indicator solution:

Methyl Red Test

(MR Test)

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Methyl Red Test (MR Test) 129

• Incubate at 37oC for 48 hours

• Add five drops of the methyl red reagent

• Mix and read immediately

• Positive tests are bright red

• Negative tests are yellow

Result

Bright red Positive (e.g Escherichia coli)

Yellow Negative (e.g Klebsiella spp)

Figure 33.1: MR test (For colour version, see Plate 9)

This test demonstrates the ability of certain bacteria to decomposethe amino acid Tryptophan to Indole When a bacterium is grown inmedium containing Tryptophan (Such as peptone water), it breaksdown the Tryptophan producing indole This indole when combinedwith a compound called para dimethyl amino benzaldehyde (added

in the form of Kovacs reagent) gives a pink coloured compoundcalled Rose Indole (Figs 34.1 and 34.2)

Hydrochloric acid (Concentrated) : 1 litre

Dissolve the aldehyde in the alcohol and slowly add the acid.Prepare in small quantities and store in the refrigerator Shake gentlybefore use

Indole Test

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Indole Test 131

Figure 34.1: Indole test (tube method)

Figure 34.2: Indole spot test

132 Practical Manual of Medical Microbiology

Method

• Inoculate peptone water medium with test organism and incubatefor 48 hours at 37oC

• Add 0.5 ml Kovac’s reagent and shake gently

• A red color in the alcohol layer indicates a positive reaction

Result

Pink colour Indole positive bacteria – Escherichia coli

No colour Indole negative bacteria – Klebsiella spp.

Note

The test can also be conducted by inserting a paper (dipped in thereagent) at the mouth of the test tube containing over night peptonewater culture of the test organism A pink colour develops in tip ofthe filter paper on indole production

This test is useful to differentiate Streptococcus pneumoniae (Pneumococcus) from viridans Streptococci Pneumococcus Colonies are soluble in bile and bile salts and viridans Streptococci are insoluble.

1 Sodium deoxycholate (100 gm/lit, 10% W/V)

2 Physiological saline (sodium chloride, 8.5 gm/lit)

134 Practical Manual of Medical Microbiology

Procedure

• Emulsify several colonies of the test organism in a tube containing

2 ml of sterile physiological saline, to give a turbid suspension

• Divide the organism suspension between two tubes

• To one tube, add 2 drops of the sodium deoxycholate reagent andmix

• To the other tube, add 2 drops of sterile distilled water and mix

• Leave both tubes for 10-15 minutes

• Look for a clearing of turbidity in the tube containing the sodiumdeoxycholate

Result

Clearing of turbidity Test organism may be Pneumococci

No clearing of turbidity Test organism is probably not

This test is used to differentiate Staphylococcus aureus which produces the enzyme coagulase, from Staph epidermidis and Staph.

saprophyticus which do not produce coagulase.

PRINCIPLE

Coagulase is an enzyme produced by Staphylococcus aureus It

converts fibrinogen to fibrin and causes clotting of plasma

Two types of coagulase are produced by most strains of Staph

Bound coagulase (Clumping factor) converts fibrinogen to fibrinwithout the help of coagulase reacting factor It can be detected bythe clumping of bacterial cells in the slide coagulase test

Coagulase Test

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136 Practical Manual of Medical Microbiology

Procedure and Results of Slide Test (to detect bound lase)

coagu-• Place a drop of saline on a slide

• Place a drop of saline on second slide also

• Emulsify a colony of the test organism in each of the drops tomake two thick suspensions

• Add a drop of undiluted plasma to one of the suspensions, andmix gently Look for clumping of the organisms within 10seconds

• No plasma is added to the second suspension This is used todifferentiate any granular appearance of the organism from truecoagulase clumping

Result

Clumping within 10 seconds Coagulase positive Staphylococci

No clumping within 10 seconds Bound coagulase absent.

Tube Test: (to detect free coagulase)

• Dilute the plasma 1 in 10 in physiological saline (mix 0.2 ml ofplasma with 1.8 ml of Saline)

Figure 36.1: Tube coagulase test

• Inoculate Staph.aureus colony in to ‘P’ test tube.

• Incubate the 3 tubes at 37 oC Examine for Clotting after 1 hour If

no clotting has occured, examine at 30 minute intervals for uptosix hour

• When, looking for clotting, gently tilt each tube If there is clotting

a jelly like mass is formed

Result

Fibrin clot Staphylococcus aureus

No fibrin clot No free coagulase produced

Antibiotic susceptibility testing has become a very essential step forthe proper treatment of infectious diseases It is used

1 To guide the clinician in selecting the best antimicrobial agent

2 To accumulate epidemiological information on the resistance ofmicroorganisms of public health importance

The choice of drugs used in a routine antibiogram is governed byvarious considerations since only a few antimicrobial agents can betested (Fig 37.1) Table 37.1 suggests the drugs to be tested in varioussituations The drugs in Table 37.1 are divided into two sets Set 1includes drugs that are available in most hospitals and for whichroutine testing should be carried out for every strain Tests for drugs

in set 2 are to be performed only at the special request of thephysician, or when the causative organism is resistant to the first-choice drugs, or when other reasons (allergy to a drug, or itsunavailability) make further testing justified

Antimicrobial Susceptibility Testing

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Antimicrobial Susceptibility Testing 139

Table 37.1: Basic sets of drugs for routine susceptibility tests

Set 1 Set 2 Staphylococcus Benzyl penicillin Gentamicin

Oxacillin Amikacin Erythromycin Co-trimoxazole Tetracycline Clindamycin Chloramphenicol

Enterobacteriaceae Sulfonamide Norfloxacin

Urinary Trimethoprim Chloramphenicol

Co-trimoxazole Gentamicin Ampicillin

Nitrofurantoin Nalidixic acid Tetracycline Blood and tissues Ampicillin Cefuroxime

Chloramphenicol Ceftriaxone Cotrimoxazole Ciprofloxacin Tetracycline Piperacillin Gentamicin Amikacin Pseudomonas aeruginosa Piperacillin Amikacin

Gentamicin Tobramycin

Figure 37.1: Antibiogram

Antimicrobial susceptibility tests measure the ability of anantibiotic or other antimicrobial agent to inhibit bacterial growth invitro This ability may be estimated by either the dilution method orthe diffusion method The common method used in diagnosticmicrobiology laboratories is modified Kirby-Bauer method

140 Practical Manual of Medical Microbiology

MODIFIED KIRBY-BAUER METHOD

Requirements

Mueller-Hinton Agar

1 Mueller-Hinton agar should be prepared from a dehydrated baseaccording to the manufacturer’s recommendations The mediumshould be such that with standard strains, zone sizes within theacceptable limits are produced It is important not to overheat themedium

2 Cool the medium to 45-50oC and pour into plates Allow to set on

a level surface, to a depth of approximately 4 mm A-9 cm diameterplate requires approximately 25 ml of the medium

3 When the agar has solidified, dry the plates for immediate usefor 10-30 minutes at 36oC by placing them in an upright position

in the incubator with the lids tilted

4 Any unused plates may be stored in a plastic bag, which should

be sealed and placed in a refrigerator Plates stored in this waycan be kept for two weeks

5 In order to ensure that the zone diameters are sufficientlyreliable for testing susceptibility to sulfonamides and co-trimoxa-zole, the Mueller-Hinton agar must have low concentrations ofthe inhibitors thymidine and thymine Each new lot of Mueller-Hinton agar should therefore be tested with a control strain ofEnterococcus faecalis (ATCC 29212 or 33186) and a disc of co-trimoxazole A satisfactory lot of medium will give a distinctinhibition zone of 20 mm or more that is essentially free of hazygrowth or fine colonies

6 For testing the susceptibility of fastidious organisms, 5% bloodshould be added to the Mueller-Hinton agar base

Antibiotic Discs

Any commercially available discs with the proper diameter andpotency can be used Stocks of antibiotic discs should preferably bekept at 20oC, or the freezer compartment of a home refrigerator isconvenient A small working supply of discs can be kept in therefrigerator for up to one month On removal from the refrigerator,

Antimicrobial Susceptibility Testing 141

the containers should be left at room temperature for about one hour

to allow the temperature to equilibrate This procedure reduces theamount of condensation that occurs when warm air reaches thecold container

Turbidity Standard

Prepare the turbidity standard by pouring 0.6 ml of a 1% (10 gm/L)solution of barium chloride dihydrate into a 100 ml graduatedcylinder, and filling to 100 ml with 1% (10 ml/L) sulphuric acid.The turbidity standard solution should be placed in a tube identical

to the one used for the broth sample It can be stored in the dark atroom temperature for six months, provided it is sealed to preventevaporation

Swabs

A supply of cotton wool swabs on wooden applicator sticks should

be prepared These can be sterilized in tins, culture tubes, or onpaper, either in the autoclave or by hot air oven

Method

To prepare the inoculum from the primary culture plate, touch with

a loop the tops of each of 3-5 colonies, of similar appearance, of theorganism to be tested

When the inoculum has to be made from a pure culture, a loopful

of confluent growth is similarly suspended in saline Inoculum fromcolonies of streptococci cannot be made by emulsification Hence,with streptococci, after inoculation the culture tubes are incubated for4-6 hours to get uniform turbidity which should be matched withthe turbidity standards

Compare the tube with the turbidity standard and adjust thedensity of the test suspension to that of the standard by adding morebacteria or more sterile saline Proper adjustment to the turbidity ofthe inoculum is essential to ensure that the resulting lawn of growth

is confluent or almost confluent

142 Practical Manual of Medical Microbiology

Inoculate the plates by dipping a sterile swab into the inoculum.Remove excess inoculum by pressing and rotating the swabs firmlyagainst the side of the tube above the level of the liquid

Streak the swab all over the surface of the medium three times,rotating the plate through an angle of 60o after each application.Finally, pass the swab round the edge of the agar surface Leave theinoculum to dry for a few minutes at room temperature with the lidclosed The antibiotic discs may be placed on the inoculated platesusing a pair of sterile forceps

A sterile needle tip may also be used to place the antibiotic discs

on the plate Alternatively, an antibiotic disc dispenser can be used

to apply the discs to the inoculated plate

A maximum of seven discs can be placed on a 9-10 cm diameterplate Six discs may be spaced evenly, approximately 15 mm fromthe edge of the plate, and one disc placed in the centre of the plate.Each disc should be pressed down gently to ensure even contactwith the medium

The plates should be placed in an incubator at 35oC within 30minutes of preparation Temperatures above 35oC invalidate theresults for oxacillin/ methicillin

Do not incubate in an atmosphere of carbon dioxide

After overnight incubation, the diameter of each zone (includingthe diameter of the disc) should be measured and recorded in mm.The results should then be interpreted according to the criticaldiameters by comparing with standard tables (Table 37.2)

The measurements can be made with a ruler on the under surface

of the plate without opening the lid

The end-point of inhibition is judged by the naked eye at theedge where the growth starts, but there are three exceptions:

1 With sulfonamides and co-trimoxazole, slight growth occurswithin the inhibition zone; such growth should be ignored

2 When β-lactamase producing staphylococci are tested againstpenicillin, zones of inhibition are produced with a heaped-up,clearly defined edge; these are readily recognizable whencompared with the sensitive control and regardless of the size ofthe zone of inhibition, they should be reported as resistant

Antimicrobial Susceptibility Testing 143

3 Certain Proteus spp may swarm into the area of inhibition aroundsome antibiotics, but the zone of inhibition is usually clearlyoutlined and the thin layer of swarming growth should be ignored

Intermediate susceptibility covers two situations It is applicable tostrains that are "moderately susceptible" to an antibiotic that can beused for treatment at a higher dosage because of its low toxicity orbecause the antibiotic is concentrated at the focus of infection (e.g.urine) The term also applies to those strains that are susceptible to

a more toxic antibiotic that cannot be used at a higher dosage In thissituation the intermediate category serves as a buffer zone betweensusceptible and resistant

Resistant: This term implies that the organism is expected not torespond to a given drug, irrespective of the dosage and the location

of the infection

For testing the response of staphylococci to benzylpenicillin, onlythe categories ‘susceptible’ and ‘resistant’ (corresponding to theproduction of b-lactamase) are recognized Staphylococci that areresistant to methicillin or oxacillin are also resistant to otherpenicillins or cephalosporins even though they show a zone ofinhibition against these drugs

144 Practical Manual of Medical Microbiology

Zone diameters, to the nearest whole mm for variousantimicrobial agents with disc content specified for each one forinterpretation as susceptible, intermediate and resistant are given

in Table 37.2

Table 37.2: Quality control – Susceptibility of control strains

Zone diameter of inhibition (mm) Antibiotic Disc potency S.aureus E coli P aeruginosa

(IU or µg) (ATCC (ATCC (ATCC

25923) 25922) 27853) Amikacin 30 20-26 19-26 18-26

Quality Assurance in Susceptibility Test

The precision and accuracy of the test are controlled by the paralleluse of a set of control strains, with known susceptibility toantimicrobial agents These quality control strains are tested usingexactly the same procedure as for the test organisms The zone sizesshown by the control organisms should fall within the range ofdiameters given in Table 37.3 When the results regularly fall outsidethis range, they should be regarded as evidence that a technical errorhas been introduced into the test, or that the reagents are at fault Eachreagent and each step in the test should then be investigated until thecause of the error has been found and eliminated

Antimicrobial Susceptibility Testing 145

The quality assurance programme should use standard referencestrains of bacteria that are tested in parallel with the clinical culture.They should preferably be run every week, or with every fifth batch oftests, and in addition, every time that a new batch of Mueller-Hintonagar or a new batch of discs is used The standard strains are:Staphylococcus aureus (ATCC 25923)

Escherichia coli (ATCC 25922)

Pseudomonas aeruginosa (ATCC 27853)

Culture for day-to-day use should be grown on slants of nutrientagar (tryptic soya agar is convenient) and stored in a refrigerator.These should be subcultured onto fresh slants every two weeks

Quality Assurance in Antibiotic Susceptibility Testing

Use antibiotic discs of 6 mm diameter

Use correct content of antimicrobial agent per disc

Stock the supply of antimicrobial discs at 20 oC

Use Mueller-Hinton medium for antibiotic sensitivitydetermination

Use appropriate control cultures

Use standard methodology for the test

Use coded strains from time to time for internal quality control.Keep the antibiotic discs at room temperature for one hour beforeuse

Incubate the sensitivity plates for 16-18 hours before reporting.Incubate the sensitivity plates at 35 oC

Space the antibiotic discs properly to avoid overlapping ofinhibition zone

Use inoculum size that produces near confluent growth.Ensure an even contact of the antibiotic disc with the inoculatedmedium

Measure the zone sizes precisely

Interpret the zone sizes by referring to standard charts

146 Practical Manual of Medical Microbiology

Table 37.3: Zone sizes with different antimicrobial agents

Antimicrobial When testing Disc Zone diameter nearest whole mm agent against content Resistant Intermediate Susceptible

Other Gram negatives 75 µg <17 18-20 >21 Nafcillin Staphylococci 1 µg <10 11-12 >13 Oxacillin Staphylococci 1 µg <10 11-12 >13 Penicillin Staphylococci 10 units <28 >29

Enterococci 10 units <14 >15 Piperacillin Pseudomonas 100 µg <17 >18

Other gram negatives 100 µg <17 18-20 >21 Ticarcillin Pseudomonas 75 µg <14 >15

Other gram negatives 75 µg <14 15-19 >20

βββββ-LACTAM/βββββ-LACTAMASE INHIBITOR COMBINATIONS

Amoxycillin/ Staphylococci 20/10 µg <19 >20 clavulanic acid

Other organisms 20/10 µg <13 14-17 >18 Ampicillin/ Staph and gram -ve 10/10 µg <11 12-14 >15 sulbactam

Piperacillin/ Pseudomonas 100/10 µg <17 >18 tazobactam

Other gram negatives 100/10 µg <17 18-20 >21 Staphylococci 100/10 µg <17 >18 Ticarcillin/ Pseudomonas 75/10 µg <14 >15 clavulanic acid

Other gram negatives 75/10 µg <14 15-19 >20 Staphylococci 75/10 µg <22 >23

Antimicrobial Susceptibility Testing 147

Note: For Vibrio cholerae, the results of disc diffusion tests for ampicillin, tetracycline and

trimethoprim/sulfamethoxazole correlate with results obtained by broth microdilution methods.

It is an agglutination test for, detection of agglutinins (Brucella) inpatient’s serum Agglutinins detected are usually either lgM or lgG

Materials Required

• Antigen: It is available from Izat Nagar or from CPHL, London or

it can be prepared from a Brucella abortus strain procured fromInstitute of Veterinary and Preventive Medicine, Ranipet,

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• Appropriate antigen control is also taken.

• Results are read by gentle agitation of the deposit and if thesupernatant is clear then it is taken as positive

• The end point or titre is the highest dilution of serum causingagglutination

• Significant titre is 100 IU or above

• H agglutinins tend to persist longer than O agglutinins Personimmunised with TAB vaccine may show high titres of antibodies

to all the antigens and so only a marked rise in titre is consideredsignificant

Brucella Agglutination Test 149

It is a neutralisation test used measure the ability of a patient’s serum

to neutralise the erythrocyte lysing capability of streptolysin O, an

extracellular enzyme produced by Streptococcus pyogenes during

infection

Requirements

• Antigen: Streptolysin O is available commercially It is reconstituted

before use

• ASO buffer: Phosphate buffered saline.

• 5% suspension of RBCs in ASO buffer: Human blood group ‘O’

or rabbit erythrocytes

• Water bath

Procedure

• Serum dilution: In a master dilution tube serum is diluted 1:100 by

adding 0.05 ml of serum to 4.95 ml of ASO buffer Subsequentdilutions are made by removing 1 ml of diluted serum from master

Anti-Streptolysin O

(ASO) Test

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Anti-Streptolysin O (ASO) Test 151

dilution tube and adding to another tube in a rack Then againadd 1 ml of ASO buffer in master dilution tube mix and remove 1

ml of this add to the second tube in the rack Similarly keep onadding 1 ml of buffer each time in master dilution tube and makerest of the dilutions The dilutions in different serum tubes will be1:100, 125, 195, 244, 305, 381, 476 and 596

• Then add 0.5 ml of the antigen to all the tubes and incubate at

• Serum should be inactivated at 56oC for 30 minutes before Use

• Rise in ASO is detectable after 1 week of infection and very hightitres are observed between third and fifth week

• False positive results are due to increase levels of lipoproteins in

serum or Contamination of serum by Staph aureus or

Pseudomonas species or by oxidation of ASO

• Commercial kits available for this are Ortho ASO and RapitexASL They are latex agglutination tests

proteins, levels over 100 mg/l frequently being found.

Thus CRP is a sensitive marker of inflammation and is a usefulindicator of the extend of activity in such disorders as rheumatoidarthritis and systemic lupus erythematosus It is also useful indifferentiating bacterial infections from viral infection as CRP levelsare elevated only in the former

Principle

CRP Screen is based on the principle of latex agglutination TheCRP Latex reagent contains uniform sized latex particles coatedwith Anti-human C-Reactive protein

CRP Screen Latex

Agglutination Slide Test

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CRP Screen Latex Agglutination Slide Test 153

When a drop of CRP Latex Reagent is mixed with a drop ofserum specimen, the latex particles agglutinate to give clearagglutination visible to the naked eye if the concentration of CRP in

the serum specimen is more than or equal to 6 mg/l.

Requirements

1 Reagent-1 (CRP Latex reagent)

2 Reagent-2 (Positive control serum)

3 Reagent-3 (Negative control serum)

4 Glass slide

5 Disposable mixing sticks

6 Disposable plastic droppers

Discard contaminated or hemolysed sera

No inactivation of the specimen is required

4 Tilt the slide back and forth slowly for 3 minutes and watch foragglutination of latex particles

154 Practical Manual of Medical Microbiology

When the test is performed with the positive control, clearagglutination of latex particles is observed within 3 minutes.When the test is performed with the negative control, noagglutination of latex particles is observed within 3 minutes

NOTES

1 Nonspecific positive result may be observed if markedly lipemic,hemolytic or contaminated serum specimen is used Use ofplasma may also give nonspecific positive results

CRP Screen Latex Agglutination Slide Test 155

2 Slight granularity of latex particles observed occasionally shouldnot be misinterpreted as a positive test result

3 All reagents of human origin were found negative when testedfor HBsAg and HIV anitbody Handle as if capable of transmittinginfection

4 Avoid contact of the reagents with skin and mucous membrane

as these contain sodium azide as preservative

5 Occasional agglutinations produced after 4 minutes have nodiagnostic significance

6 Rheumatoid factors in the sample may also agglutinate the latexreagent

7 A prozone phenomenon appears at 80 ug/ml

Veneral disease research Laboratory in USA developed this slideflocculation test for the lab diagnosis of Syphilis This is a non-specific standard test for syphilis using non-specific, non-treponemalantigen- cardiolipin, for the detection of antibodies

It is a simple, rapid convenient and economical procedure forseriological testing for syphilis It has high sensitivity and specificityand can be used for rapid and exact quantitative titration of reactiveserum samples It is well suited for mass serologic surveys and forthe examination of large number of serum samples in STD clinics.Nowadays of modification of VDRL test known as RPR (RapidPlasma reagin) test is used in laboratories for rapid diagnosis.VDRL antigen can be obtained from Laboratories of Serologists,Calcutta, a Government of India Organisation

Requirments

1 VDRL antigen

2 Glass slide 2 × 3” with 12 paraffin rings approxiamtely 14 mm indiameter (Similiar slides with permanently fixed ceramic ringsare commercially available)

VDRL Test

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on to the saline while rotating the bottle on a flat surface.

The Antigen is added drop by but rapidly so that it takesapproximately 6 seconds to complete the delivery Blow the lastdrops of the antigen and continue rotation of the bottle for 10 moreseconds Then add 4.1 ml of buffered saline from a 5.0 ml pipette.Stopper the bottle and shake it vigorously for approximately 10seconds

This emulsion is kept for 30 minutes for maturation It can beused only within 24 hours This quantity is sufficient for 250 serumtest

Preparation of Serum

Clear serum obtained from centrifuging whole clotted blood of thepatient is heated for 30 minutes at 56oC This is to inactivate theComplement activity of the serum which may interfere the reaction

Preliminary Testing of Antigen Emulsion

Each preparation of antigen emulsion should first be examined bytesting known reactive and non-reactive sera An unsatisfactoryantigen emulsion should not be used