tests

1. Department of MICROBIOLOGY
U.P. Pt. Deen Dayal Upadhyay Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Ansundhan
Sansthan,DUVASU Mathura
Presentation on
Biochemical tests &
Serological tests for
Bacterial Identification
Suggested by :
Dr. Sharad
Dr. Ajay Pratap Singh
Dr. Ruchi Tiwari
Dr. Vinod kumar
Presented by :
Harshit Saxena
B.V.Sc & A.H.
Enroll. No. – V-1598/16

2. Diagnosis of a
infectious disease
A Clinician traces following path in diagnosing a disease:
1. History of Patient: By owner; Herd manager
↓
2.Signs & Symptoms: close external examination
↓
3.Isolation(if possible) & Identification of pathogen & its
reactions in body

3. Bacterial
Identification
Cultural
Characterstics
Morphological
Test
Biochemical
Tests
Immunological
Tests
Molecular
Tests
Basis

4. 1. Urease Test
Purpose : Specifically- Differentiating between Proteus & Non Lactose
Fermenting enteric Bacteria (Salmonella & Sheigella)
Principle : Few bacteria undergo following reaction by Urease
Enzyme prodution
Urea + water → Ammonia + Carbondioxide + Water
Ammonia increase of pH → Colour of Phenol Red indicator changes
from orange red to deep pink or purplish red cerise
Materials : Cultures of Pseudomonas fluorescens & Proteus vulgaris
4 urea broth tubes
Inoculation loop
Marker, Bunsen burner
Urease

5. Method : Inoculate urea broth tubes with test bacteria
↓
Mix well & incubate at 37°C for 24-48 hrs
↓
Observe Change colour
Interpretation& Result: Tubes inoculated with Proteus vulgaris/
Urease Positive bacteria – Deep pink reaction (Positive Reaction)
Tubes inoculated with Pseudomonas fluorescence no change in
colour of broth(Negative Reaction)

6. 2.Nitrate Reduction Test
Purpose : Differentiation of members of Enterobacteriaceae from
other bacteria
Principle :
E. coli : Reduce nitrate to nitrite
Pseudomonas : Reduce completely to
molecular Nitrogen
S. aureus : Unable to reduce nitrate
Bacterial broth having 0.5% KNO3
↓
After incubation seen for gas CO2 & NO2
production(from nitrate reduction & citric acid cycle)
↓
By addition of 1. Sulfanilic acid
2. N,N di-CH3-1 naphthalamine
↓
Pink or Red colour produced due to nitrite in
medium

7. Materials: Culture of test bacteria, little soil, Nitrate broth tubes
Nitrate test reagent A
Nitrate test reagent B
Nitrate test reagent C or Zn powder
Test tubes
Procedure& Interpretation: Label bacterial tubes, last one is with Garden soil, 5th as control
↓
Inoculate respective bacterial tubes accordinly with different bacteria, fourth one with garden
soil
↓
Incubate all TT at 37°C for 24-48 hrs & observe after incubation for growth, control no growth
↓
In all TT add 0.5ml each of Nitrate Reagent A&B
↓
Development of RED colour – POSITIVE Reaction, no colour in control
↓
Confirm negative test by adding Nitrate C Reagent-5-10 drops or adding Zn powder
↓
Colour turn red in 5-10min if NO3 is present in medium i.e, Negative NO3 reduction test

8. Experimental observation

9. Catalase Test
Purpose: Differentiate ‘Catalase’ enzyme producing(generally
AEROBIC) & Non producing bacteria
Differentiate Staphylococcus sp.(+test) from Stretococcus sp.(- test)
Principle: Catalase enzyme protect bacteria from destruction by H2O2
produced in Aerobic metabolism & release O2
2H2O2 2H2O + O2
Material: H2O2 , Inoculation loop, bacterial culture, Glass slides
Catalase

10. Procedure: Septically with sterile inoculation loop take bacterial
culture from petri plate to glass slide
↓
With pipette add 2-3 drops of H2 O2
Interpretation & Result:
Immediate appearance of Gas bubbles : POSITIVE Test
Absence of gas bubbles : NEGATIVE Test
Experimental observation

11. Lipid Hydrolysis Test
Purpose: Detect lipase producing ability of Bacteria detecting
pathogenicity of a bacteria
Enterobacteriaceae, Fusobacterium, Propionibacterium, Clostrid
ium,Pseudomonas, Mycoplasma, Corynebacterium,
and Staphylococcus give + test
Principle: Organism producing exoenzyme ‘lipase’ hydrolyses
Tributyrin oil in Tributyrin plates ( others oils too Corn oil,
soyabean oil, egg yolk etc)
Producing clear halo – Zone of hydrolysis in plates
Materials: Tributyrin agar plates
Culture of Salmonella typhimurium
Culture of Pseudomonas aeruginosa
Inoculation loop

12. Procedure:
Divide Tributyrin plate using marker in 2 halves
↓
Write P.aeruginosa on one and S.typhimurium on other side
↓
Streak respective bacterial culture on the plate
↓
Incubate at 30°C for 24-48 hours
Interpretation & Result:
Halo zone around streak of P.aeruginosa – POSITIVE Test
No zone of clearence - NEGATIVE Test

13. Oxidase Test
Purpose:To detect wheather a organism is Aerobe or not
Oxidase Positive Organisms: Pseudomonas, Neisseria, Alcaligens,
Aeromonas, Campylobacter, Vibrio, Brucella, Pasteurella, Moraxella,
Helicobacter pylori, Legionella pneumophila, etc.
Oxidase Negative Organisms: Enterobacteriaceae
Principle Detect whether the sample bacteria produces Cytochrome
c oxidase enzyme by:
N,N,N,N-tetramethyl-p-phenylenediamine(TMPD) a redox indicator
a. Appear blue when oxidised
b. Colourless when reduced
Materials: Culture of Pseudomonas aeruginosa,E.coli
Discs impregnated with TMPD solution
Inoculation loop

14. Procedure:
Take a impregnated disc over a glass slide
↓
Moisten it
↓
Take bacterial culture from petri plate or broth and place on the
disc
Interpretation & Result:
a. Disc turn dark purple : POSITIVE Test –Oxidising bacteria
(here P.aeruginosa
b. No change : NEGATIVE Test – Reducing bacteria
Experimental observation

15. DNase Production Test
Purpose: To detect DNase producing bacteria attributed to
Pathogenicity; Used when serum is not available to test
Principle : DNase culture contain nutrients for bacteria, DNA &methyl
green indicator
Methyl green a cation that binds → Negatively charged DNA
DNase breaks the DNA → No binding to indicator → Production of
clear halo
Materials :
DNase agar plate, Staph aureus/E.coli & Serratia. marcescens culture
1N HCl
Inoculation loop, Pipette

16. Method:
Divide DNase culture plate my marker to 2 halves mark the name
of 2 bacteria
↓
With sterile Inoculation loop streak heavily the respective bacterial
cultures ; Incubate at 37°C for 24-48 hrs
↓
Flood it with 1N HCl with pipette
Interpretation & Result:
a. Zone of clearence around streak of S. marcesens show DNase
activity & POSITIVE Test
b. No clearance: NEGATIVE Test

17. Starch hydrolysis or Amylase Production Test
Purpose: To see if the microbe can use starch as a source of
carbon and energy for growth & produce Amylase;Bacillus
subtilis and Escherichia coli, is compared on starch agar.
Principle: Exoenzyme Amylase produced by Bacteria hydrolyses
starch to Dextrins Maltose & Glucose
Addition of Iodine tells amount of satrch hydrolysed by bacteria
Starch Dextrins+Maltose+Glucose
Materials:
Test Bacteria (Bacillus subtilis & Escherichia coli)
Starch Agar media
Gram’s iodine soln.
Petri plates, Inoculation loop
Alpha- Amylase + H2O

18. Procedure:
Take Starch Agar plates mark according to bacterial culture to be
inoculated
↓
Using inoculation loop singly streak with respective bacteria;
incubate at 37°C for 24-48 hrs
↓
Flood the plate with I2 soln.
Interpretation & Result:
Clear Zone around bacterial colonies : POSITIVE Test
Dark blue colouration around bacterial colonies : NEGATIVE Test
Experimental observation

19. Gelatin hydrolysis(Gelatinase production)
Test
Purpose: Helpful in differentiating species of
Bacillus, Clostridium, Proteus, Pseudomonas, and Serratia;
Gelatinase-positive, pathogenic Staphylococcus aureus from the
Gelatinase-negative, nonpathogenic S. epidermidis .
Principle: Gelatin solidify at temperature below 28 °C this property
loses when gelatinase enzyme produced by bacteria act on gelatin
Materials: Bacterial culture of Bacillus subtilis, E.coli, Proteus vulgaris
12% Gelatin tubes

20. Method:
Take tubes containing gelatin with nutrient media
↓
Inoculate them heavily with test bacteria using Stab method
↓
Incubate for 48 hrs then place in refrigerator for 30 min.
Interpretation & Result:
Observe the tubes
a. Media is still intact –NEGATIVE Test
b. Media loses & gelatin hydrolyses to liquid media – POSITIVE
test

21. # Period of incubation can be extended for 2 weeks as some
bacteria produces Gelatin in very small quantities

22. IMVIC Tests
1.Indole Test
2.Methyl Red Test
3.Voges-Proskauer Test
4.Citrate Test
Purpose: For Identification of bacteria in
family ‘Enterobacteriaeae(Enterics)’

23. Indole Test
Purpose: To detect bacteria producing Tryptophanase enzyme
Principle: Some bacteria oxide EAA –
Tryptophane Indole+Pyruvic Acid +Ammonia
Indole + Kovac’s Reagent Rosindole (Cherry Red
(p-dimethylaminobenzaldehyde) Complex)
Materials:
E.coli & Enterobacter aerogens cultures in NB
1%tryptone broth in 3 test tubes
Kovac’s Reagent
Inoculation loop, Bunsenburner, Pipette
HCl Butanol
Tryptophanase

24. Procedure:
1% Peptone water in 3 test tubes 5ml each
↓
Inoculate the 2 test tube with 2 bacterial cultures separately
Incubate at 37°C for 24-48 hrs
↓
After 48 hours add 1ml kovac’s Reagent shake 15-20min with time gap
↓
Keep the tubes let reagent react to the surface
Interpretation & Result:
Development of Cherry red colour (E.coli) – POSITIVE Test
No colour production- NEGATIVE Test

25. Methyl Red & Voges Proskauer Test
•Clark and Lubs developed MR-VP Broth
•Both the MR and VP tests performed from the same inoculated
medium by aliquoting portions to different tubes
•Bacteria incubated in MR-VP broth having Glucose & Peptone
Methyl Red Test
Purpose: Originally used to distinguish between members of the
family Enterobacteriaceae
• But now also used to characterize other groups of bacteria including
Actinobacteria
Principe: Some bacteria have ability to :
•Glucose Lactic Acid +Acetic Acid+Formic Acid
Mixed Acid Pathway

26. •Acid so produced decreases the pH to 4.5 or below, which is
indicated by a change in the colour of methyl red from yellow to
red
Materials:
MRVP broth (pH 6.9)
Ingredients per liter of deionized water:
buffered peptone= 7.0 gm
glucose= 5.0 gm
dipotassium phosphate= 5.0 gm
Methyl red solution, 0.02%
a. Dissolve 0.1 g of methyl red in 300 ml of ethyl alcohol, 95%.
b. Add sufficient distilled water to make 500 ml.
c. Store at 4 to 8 degree C in a brown bottle. Solution is stable for
1 year.

27. Procedure:
Prior to inoculation, allow medium to equilibrate to room temperature
↓
Using organisms taken from an 18-24 hour pure culture, lightly inoculate
the medium
↓
Incubate aerobically at 37 degrees C. for 24 hours
↓
Following 24 hours of incubation, aliquot 1ml of the broth to a clean TT
↓
Reincubate the remaining broth for an additional 24 hours
↓
Add 2 to 3 drops of methyl red indicator to aliquot
Result & Interpretation:
Positive Reaction: A distinct red color (A)Examples: E. coli, Yersinia sps,
Negative Reaction: A yellow color (B)
Examples: Enterobacter aerogenes, Klebsiella pneumoniae, etc

28. https://microbiologyinfo.com/category/biochemical-test/

29. Voges Proskauer Test
Principle: Used to determine if an organism produces acetylmethyl
carbinol from glucose fermentation
Acetylmethyl carbinol is converted to diacetyl in the presence of ∝-
naphthol, strong alkali (40% KOH), and atmospheric oxygen
The ∝-naphthol act as a color intensifier
The diacetyl and quanidine-containing compounds in the peptones of
the broth condense : pinkish red polymer

30. Materials:
Ingredients per liter of deionized water:
buffered peptone= 7.0 gm
glucose= 5.0 gm
dipotassium phosphate= 5.0 gm
Voges-Proskauer Reagent A: Barritt’s reagent AAlpha-Naphthol, 5%50
gmAbsolute Ethanol1000 ml
Voges-Proskauer Reagent B: Barritt’s reagent BPotassium Hydroxide400
gmDeionized Water1000 ml
Procedure:
Prior to inoculation, allow medium to equilibrate to room temperature
↓
Using organisms taken from an 18-24 hour pure culture, lightly inoculate
the medium

31. Incubate aerobically at 37 degrees C. for 24 hours
↓
Following 24 hours of incubation, aliquot 2 ml of the broth to a clean test
tube
↓
Re-incubate the remaining broth for an additional 24 hours
↓
Add 6 drops of 5% alpha-naphthol, and mix well to aerate
↓
Add 2 drops of 40% potassium hydroxide, and mix well to aerate
↓
Shake the tube vigorously during the 30-min period
Interpretation & Result:Positive Reaction: A pink-red color at the surface
Examples: Viridans group streptococci (except Streptococcus
vestibularis), Listeria, Enterobacter, Klebsiella, Serratia marcescens,
Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc

32. Negative Reaction: A lack of a pink-red color
Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia,
Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio
vulnificus, and Vibrio parahaemolyticus etc.
https://microbiologyinfo.com/voges-proskauer-vp-test-principle-reagents-procedure-and-
result/

33. Citrate Test
Purpose: Citrate utilising/fermenting bacteria are identified
List of Bacteria which gives positive citrate utilization test
Klebsiella pneumoniae.
Enterobacter species (minority of strains gives negative result)
Citrobacter freundii.
Salmonella other than Typhi and Paratyphi A.
Serratia marcescens.
Proteus mirabilis (minority of strains gives negative result)
Principle: Bacteria that can grow on Simmon’s citrate Agar(SCA)
produce an enzyme, citrate-permease, capable of converting
citrate to pyruvate
Bacteria metabolize citrate, the ammonium salts are broken down
to ammonia, which increases alkalinity

34. The shift in pH turns the bromthymol blue indicator in the medium
from green to blue above pH 7.6
Materials: E.coli & Enterobacter aerogens culture in NA
Simmon’s citrate agar plates
Inoculation loop & Needle
Method:
Inoculate SCA with bacterial cultures one kept as control
↓
Incubate all at 37°C for 24-48 hrs
Interpretation & Result:
Growth of bacteria=Prussian blue colour develops=POSITIVE Test
No growth=Medium remains green=Negative test

36. H2S production & Motility
Purpose:Determines whether the microbe reduces :-
Sulfur-containing compounds to sulfides metabolism i.e,
Citrobacter spp; Clostridium perfringens
Principle: Reduction of compounds such as thiosulphate, sulphite,
sulphate by some bacteria produce H2 S
Materials:
•Plant cultures of Pseudomonas aerogenosa, Escherichia coli&
Proteus vulgaris
•Sulfide motility medium(SIM) tubes semi solid
•Inoculation loop & needle

37. Procedure:
Label all SIM agar tubes with name of bacteria to be tested
↓
Stab the tubes with respective bacteria incubate 37°C for 24-48 hrs
Interpretation & Result:
Proteus vulgaris tube=Blackening seen= POSITIVE Test
# Motility test = Zone of growth localised near stab(non motile) or
diffused in media(motile)

38. Carbohydrate fermentation test
Purpose:
Enterobacteriaceae family are glucose fermenters (they can
metabolize glucose anaerobically)
Maltose fermentation differentiates Proteus vulgaris (positive)
from Proteus mirabilis (negative)
Rapid carbohydrate utilization test can be performed to
identity Corynebacterium diphtheriae & other sp. Of this bacteria
Principle:
Tests for the presence of acid and/or gas produced from
carbohydrate fermentation
Basal medium containing a single carbohydrate source is used for
this purpose

39. •A pH indicator (such as Andrade’s solution) which will detect the
the lowering of the pH of the medium due to acid production
• Small inverted tubes called Durham tube immersed in the
medium to test production of the gas (hydrogen or CO2 )
Materials:
Composition of Phenol Red Carbohydrate Broth
Trypticase or proteose peptone No. 3: 10 g
Sodium Chloride (NaCl): 5 g
Beef extract (optional): 1 g
Phenol red (7.2 ml of 0.25% phenol red solution): 0.018 g
Carbohydrate source: 10 g

40. Procedure:
Prepare broth media
↓
Fill 4-5 ml of phenol red carbohydrate broth
↓
Insert a Durham tube to detect gas production.
Autoclave the prepared test media (at 121°C for 15 minutes) to
sterilize for 3 minute
The preferred carbohydrate concentration is 1%
↓
Aseptically inoculate each test tube with test microorganism
↓
Alternatively, inoculate each test tube with 1-2 drops of an 18- to
24-hour brain-heart infusion broth culture of the desired
organism
Incubate tubes at 35-37°C for 18-24 hours

41. Interpretation & Result:
Acid production:
Positive: Yellow colour
Negative: Red colour
Gas Production
Positive: Bubbles will be seen in the inverted Durham tube
Negative: No bubbles

42. Coagulase test
Purpose:To differentiate Staphylococcus aureus (positive) from
Coagulase negative sp. from Coagulase Negative (CONS)
Principle: Detects clumping factor (formerly referred as cell-
bound coagulase)
Clumping factor directly converts fibrinogen to fibrin causing
agglutination
Heavy suspension of organism is made on glass slide and mixed
with drop of plasma
Materials: Clean glass slide
Bacterial cuture to be tested
Serum, Inoculation loop

43. Procedure:
Emulsify a staphylococcal colony in a drop of water on a clean and
grease free glass slide
↓
Dip a inoculating wire into the undiluted plasma at room
temperature, withdraw, and stir the adhering traces of plasma (not
a loopful) into the suspension on the slide
↓
Flame the wire and repeat for the control suspensions
↓
Read clumping of cocci visible to the naked eye within 10 seconds
Interpretation & Result:
Coagulase positive: Macroscopic clumping in 10 second
Coagulase negative: No clumping in either drop

45. Serological
Tests

46. What is Serology & Serological Tests???
“Serology - is the science of identifying &measuring
antibody or antigen in body fluids”
Serological Tests refers to procedures applied in order to
scan either antibody of antigen in the body fluid of a
organism
Need???
•Diagnosis of a disease - 1° Use
•Checking competene
Eg: a)Transfusion
b)Feeding of new born with its Dam’s milk( IgG Ab testing)

47. Antigen Tests
Antibody Tests
•Isolation & Identification of a Pathogen
•Early diagnosis
•When Isolation & Identification of a Pathogen is not easy
•Transfusion Cases
•Early diagnosis
•Autoimmune diseases
•Checking effectivenes of treatment prescribed

48. Serological Tests Classification:
1. Primary Serological Tests
2.Secondary Serological Tests
3.Tertiary Serological Tests

49. Primary Serological Tests
Directly measure the binding of antigen and antibody
(i.e.; directly measure or visualize the immune
complex).
Most sensitive techniques
Eg:
1.Enzyme linked Immunosorbent assay (ELISA)
2. Radioimmunoassay (RIA)
3.Immnunoperoxidase Test
4.Flourescent Antibody Test
3. Western blotting

50. ELISA :
Enzyme linked
immunosorbent assay

51. History:
1971 : two Swedish scientists : Eva Engvall and Peter
Perlman invented a test that revolutionized medicine
Why ELISA is one of the most celebrated diagnostic
technique???

52. Principle:

54. Ways of performing ELISA:

55. Direct v/s Indirect ELISA

56. Apply Antigen
1. Add 100 μl antigen diluted in coating solution to appropriate wells.
2. Incubate 1 hour.
3. Empty plate and tap out residual liquid.
Block Plate
1. Add 300 μl blocking solution to each well.
2. Incubate 15 minutes, empty plate and tap out residual liquid.
Add Secondary Antibody Solution
1. Add 100 μl secondary antibody solution to each well.
2. Incubate 1 hour.
3. Empty plate, tap out residual liquid.
Wash Plate
1. Fill each well with wash solution. 2. Empty plate, tap out residual liquid.
3. Repeat 3 - 5 times.
4. Give final 5 minute soak with wash solution; tap out residual liquid.
React Substrate
1. Dispense 100 μl substrate into each well.
2. If desired, after sufficient color development add 100 μl of the appropriate stop solution to
each well.
3. Read plate with plate reader. Recommended filters:ABTS: 405-415 nm TMB-based
substrates: unstopped 620-650 nm stopped 450 nm pNPP: 405-415 nm BluePhos: 595-650 nm
FirePhos: 460-505 nm
Direct ELISA(Protocol)

57. Indirect ELISA(Protocol)
Apply Antigen
1. Add 100 μl antigen diluted in coating solution to appropriate wells.
2. Incubate 1 hour.
3. Empty plate and tap out residual liquid.
Block Plate
1. Add 300 μl blocking solution to each well.
2. Incubate 15 minutes, empty plate and tap out residual liquid.
React Primary Antibody
1. Add 100 μl secondary antibody solution to each well.
2. Incubate 1 hour.
3. Empty plate, tap out residual liquid.
Wash Plate
1. Fill each well with wash solution.
2. Empty plate, tap out residual liquid.
3. Repeat 3 - 5 times.
Add Secondary Antibody Solution
1. Add 100 μl diluted secondary antibody to each well.incubate 1 hours
2.Empty plate, tap out residual liquid and wash as above.
3. Give final 5 minute soak with wash solution; tap out residual liquid.
React Substrate
1. Dispense 100 μl substrate into each well.
2. If desired, after sufficient color development add 100 ml appropriate stop solution to each well.
3. Read plate with plate reader

58. Advantages & Disadvantage
Direct ELISA• Quick, only one antibody and fewer steps are used.
• No cross-reactivity of secondary antibody
• Immune reactivity of the primary antibody might be adversely
affected by labeling.
• Minimal signal amplification
Indirect ELISA
• Versatile as many primary antibodies can be made in onespecies
and same labeled secondary antibody can be used for detection
• Maximum immune reactivity of the primary antibody is retained
because it is not labeled
• Sensitivity is increased because each primary antibody contains
several epitopes allowing for signal amplification.

60. Sandwich ELISA-Protocol
Apply Capture Antibody :Same manner
Block Plate Same manner
React Sample Antigen
1. Add 100 μl secondary antibody solution to each well.
2. Incubate 1 hour or overnight.
3. Empty plate, tap out residual liquid.
Wash Plate
1. Fill each well with wash solution.
2. Empty plate, tap out residual liquid.
3. Repeat 3 - 5 times.
Add Secondary Antibody Solution
1. Add 100 μl diluted secondary antibody to each well.
2. Incubate 1 hour at room temperature.
3. Empty plate, tap out residual liquid and wash as above.
4. Give final 5 minute soak with wash solution; tap out residual liquid.
React Substrate
1. Dispense 100 μl substrate into each well.
2. If desired, after sufficient color development add 100 ml of the appropriate stop solution to
each well.
3. Read plate with plate reader

61. Immunoperoxidase Test
•Same as ELISA except it is used on tissue sections
•Also known as In situ ELISA
•Antibody conjugated with enzyme & when suitable
substrate added give colour seen by microscope
•Used for sections & smears that are formalin fixed thus
not use for pathogen isolation
•Detect Antigens & cellular changes caused by it

62. Microscopic Demonstration

63. Fluorescent Antibody Test
•Same as ELISA except that here Ab are conjugated to Fluorescent dye
(say Fluorescein isothiocynateTetramethl, rhodamine, Alexafluor,
Auramine)in place of enzyme
•Dye after irradiation with UV light – 290&145nm emits green light
•Most suitable for Rabies Test

64. Procedure:
Infect coverslip with Goat pox virus of different dilutions
↓
Add maintainence media incubate at 37°C for 3 days
↓
Fix the monolayers & decant the medium from both test n control
↓
Dry monolayers stain with Hyperimmune serum leave for 1hour
↓
Wash with PBS
↓
Stain monolayers with FITC conjugate leave for a hour
↓
Wash molasses mount the coverslip on glass slide with 50%
glycerine saline
Examine under fluorescent microscope

65. Radioimmune Assay
•Developed by Rosalyn Sussman Yalow, Roger Guillemin, and Andrew
Schally
• This revolutionary development earned Dr. Yalow the Nobel Prize
for Medicine in 1977
Principle & Procedure:

67. Secondary Serological Tests
Secondary binding tests are tests that detect and measure the
consequences (secondary effect) of antigen-antibody
Interaction
These consequences include:
• Precipitation of soluble antigens
• Clumping (agglutination) of particulate antigens
• Neutralization of bacteria, viruses, or toxins; and
•Activation of the complement system
•They are usually less sensitive than primary binding tests, but
may be easier to perform

68. Precipitation Tests
“Precipitation reactions are based on the interaction of two soluble
reactants antibodies and antigens that come together to make one
insoluble product”
•Antigen: Precipitinogen Antibody:Precipitin
•Presence of electrolyte & pH needed
•Produced by multivalent Ab & presence of Zonal Phenomenon
•Used for indentifying bacterial types; cross reactivity; Atg. relatedness
•Can be performed as :
Single dimension double diffusion: Oudin Test
Double dimension double diffusion: Ouchterlony Test

69. Lattice hypothesis
Multivalent Antigens react with Bivalent Antibodies - Varying
proportions-Depending on Ag-Ab ratio at zone of equivalence
Antigenic identity; Partial identity; Non Identity is also studied

70. A gel-diffusion, antibody-antigen precipitation test that depends on
simple vertical diffusion in one dimension
↓
Gel column is prepared containing a homogeneous distribution of
antibody molecules
↓
Above this is layered an aqueous suspension of antigen molecules is
poured
↓
As these formed diffuse into the gel, a moving zone of antigen-
antibody precipitates if the two are complementary : Positive Test
Oudin Test

71. Ouchterlony double immunodiffusion
a.Agar Gel Precipitation Test
Principle:
Antigen & Antibody both moves in solid agar to form insoluble
complexes & band formation
Visualization done against dark background with eluminated
object obliquely from bottom
Material used:-
1. Test sample as Antigen or Antibody
2. Known Antigen or Serum Containing known Antibodies
3. 1%Noble Agar(Difco) in PBS pH-7.4

72. 4. Staining soln. – Mixing n filtering
a. Coomsaie brilliant blue R-250
b. Glacial Acetic Acid
c. Methanol
d. Distilled Water
Procedure:
Pour 5ml molten agar in petri plates tranfer
to refrigerator at 4°C for 20minutes
↓
Punch well in Agar
↓
Remove agar from wells seal the base by 1.1% molten agar soln
↓
Fill the central well with Antigen & Periphera wells- (+)&(-) antisera with
pasteurpipettes
↓
Petri plate kept at humidified place for few hours- overnight
↓
Read pattern of precipitation line

74. b.Countercurrent Immunoelectrophoresis
•In electric field:
Antigens: Negatively charge Migrate to anode
Antibody: Positively charge Migrate to Cathode
•Endosmotic flow & Double dimension double diffusion tests
•Same as AGPT with application of concept of Elecrophoresis
•Reduce duration in 30-60 minutes Antg-Atb complexes can be
visualized
•Electrophoresis use here 0.02M Barbitone buffer pH8.6
•Migaration of Antigens can be seen by adding Bromophenol
blue dye

75. Rocket Immunoelecrophoresis Test
Used in quantification of Antigen
•Length of Precipitation are formed when Antigen is
electrophresedd into an agar layer containing antibody
•In electric field:
Antigens: Negatively charge Migrate to anode
Antibody: Positively charge Migrate to Cathode
Antigens migrate- get dilute-Ag held back in complexes-Equivalence
point reached precipitate form
More antigen – precipitate redissolves move forwards
Antigens exhaust – Stable arc forms
Height of rockets directly proportional to concentration of antigen

77. Agglutination Test
•Particulate antigen(Agglutinogen) mixed with a antibody(Agglutinin)
in presence of electrolyte(NaCl) at suitable temp & pH lead to clumping
& Agglutination
•More sensitive than precipitation
•Monovalent or incomplete antibodies not cause agglutination
•Most used test in identifying type specie
•Types
1.Slide Agglutination
2.Tube Agglutination
3.Heterophile Agglutination
4.Antiglobulin coombs test

78. Slide agglutination Test
• Suspension of unknown antigen is kept on slide and a drop of
standardized antiserum is added or vice versa
• A positive reaction is indicated by formation of visible clumps.
E.g. Widal test, RPR test.

79. Tube Agglutination Test
• Agglutination test performed in tube and standard quantitative
technique for determination of antibody titre
•In this method serum is diluted in a series of tubes and standard
antigen suspensions (specific for the suspected disease) are added
to it
•After incubation, antigen-antibody reaction is indicated visible
clumps of agglutination.

80. Heterophile Agglutination Test
This test depends on demonstration of heterophilic antibodies
in serum present in certain bacterial infection
Antiglobulin (Coombs) test:
Devised by Coombs, Mourant, and Race for detection of incomplete
anti-Rh antibodies that do not agglutinate Rh+ erythrocytes in saline
serum containing incomplete anti-Rh antibodies is mixed with Rh+
erythrocytes in saline
incomplete antibody antiglobulin coats the surface of erythrocytes but
does not cause any agglutination.
When such erythrocytes are treated with antiglobulin or Coombs
serum (rabbit antiserum against human gamma globulin), then the
cells are agglutinated.

82. Complement Fixation Test
•Detection of the presence of either specific antibody or
specific antigen in a patient's serum, based on whether complement
fixation occurs
•Process
•Serum is separated from the patient
•Patients naturally have different levels of complement proteins in their
serum.
•To negate any effects this might have on the test, the complement
proteins in the patient's serum must be destroyed and replaced by a
known amount of standardized complement proteins

83. •A)The serum is heated in such a way that all of the complement
proteins—but none of the antibodies—within it are destroyed.
•B) known amount of standard complement proteins are added to
the serum. (guinea pig serum.)
•The antigen of interest is added to the serum
•Sheep red blood cells (sRBCs) [2] which have been pre-bound to anti-
sRBC antibodies are added to the serum
• The test is considered negative if the solution turns pink at this point
and positive otherwise

85. Tertiary Binding Test
If an organism or antigen possesses biological activity, antibody can
be measured by their ability to neutralize this activity
The activities that may be neutralized include hemolysis of RBCs, lysis
of nucleated cells, and disease or death in animals
Reactions such as these are subject to high degree of variability

86. Neutrilization Test
1.In vivo –Schick’s Test
Schick test - used to determine whether or not a person is
susceptible to diphtheria (Corynebacterium diphtheria).
A small amount (0.1 ml) of diluted (1/50 MLD) diphtheria toxin
is injected intradermally into the arm of the person.
The skin around the injection will become red and swollen,
indicating a positive result.
This swelling disappears after a few days.
If the person has immunity, then little or no swelling and
redness will occur, indicating a negative result
2. In Vitro (Antistreptolysin “O” titration)

87. Test based on Type 4 Hypersensitivity
1.Tuberculin Test
2.Mallein Test
3.Johnin Test etcc..
Interadermal antigen inoculation is done and inflammatory
changes seen after 24-48 hours
Presence of inflammation is Positive Result
Absence of inflammation Negative result
# Tests based on Inoculation of test sample in live animals
also falls under the category of tertiary binding tests

88. References
1.https://www.cartercenter.org/resources/pdfs/health/ephti/library/lecture_notes/med
_lab_tech_students/serology.pdf
2.https://www.creative-diagnostics.com/ELISA-guide.htm
3.Funded under USAID Cooperative Agreement No. 663-A-00-00-0358-00.
Produced in collaboration with the Ethiopia Public Health Training Initiative, The Carter
Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education.
4. https://www.researchgate.net/figure/Schematic-of-standard-immunoassay-
procedure-either-radioimmunoassay-or-enzyme_fig2_264752239
5.Laboratory Manual of Microbiology – Vivek Kumar
5.Veterinary Immunology by Ian R.Tizard

89. Thank
You