1. BIOCHEMICAL AND SEROLOGICAL
TESTS
INTRODUCTION
• Biochemical tests are used to differentiate different organisms based on their genus and species characteristics.
• Biochemical tests are performed on pure culture.
• The following are some of the common biochemical tests used for differentiation of different bacteria.
1
1. Catalase test
This test is used to differentiate those bacteria that produce the enzyme catalase such as Staphylococci from non-catalase producing
bacteria such as Streptococci.
Principle:
– Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water.
2. 2
– An organism is tested for catalase production by bringing it into contact with hydrogen peroxide.
– Bubbles of oxygen are released if the organism is a catalase producer. The culture should not be more that 24 hours old.
1. Catalase test … Cont’d
Material Required
• Hydrogen peroxide (3% H2O2 )
•
•
Test tubes
Swab
Method
• Pour 2-3 ml of the hydrogen peroxide solution into a test tube.
•
Using a sterile wooden stick or a glass rod, remove several colonies of the test organism and immerse in the hydrogen peroxide
solution.
• Look for immediate bubbling.
– Important: Care must be taken when testing an organism cultured on a medium containing blood because catalase is present in
red cells. If any of the blood agar is removed with the organism, a false positive reaction may occur.
3. 1. Catalase test …Cont’d
Figure: Catalase tube test.
Right: Shows a positive test. Left: Shows a negative test.
3
4. 4
Results
• Active bubbling ----- Positive test-
•
Catalase produced
No release of bubbles ----- Negative test - No catalase produced
1. Catalase test …Cont’d
Note:
•if the organism has been cultured on an agar slope, pour about 1ml of the hydrogen peroxide solution over a good growth of the
organism, and look for the release of bubbles.
Control
• Positive catalase control – Staphylococcus species
• Negative catalase control – Streptococcus species
2. Coagulase Test
•This test is used to differentiate Staphylococcus aureus which produces the enzyme coagulase, from S. epidermidis and S.
saprophyticus which do not produce coagulase.
5. 5
Principle
• Coagulase causes plasma to clot by converting fibrinogen to fibrin.
•
➢
Two types of coagulase are produced by most strains of S. aureus.
Free Coagulase which converts fibrinogen to fibrin by activating a coagulase-reacting factor present in plasma.
– Free coagulase is detected by the appearance of a fibrin clot in the tube test.
➢Bound coagulase (clumping factor) is found on bacterial cell surface which converts fibrinogen directly to fibrin without
requiring a coagulase reacting factor.
– It can be detected by the clumping of bacterial cells in the rapid slide test.
• It is usually recommended that a tube test should be performed on all negative slide tests.
•
•
A tube test must always be performed if the result of the slide test is not clear, or when the slide test is negative.
Before performing a coagulase test, examine a Gram stained smear to confirm that the organism is a Gram positive coccus.
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2. Coagulase Test…Cont’d
Material Required
•EDTA anticogulated human plasma.
– The plasma should be allowed to warm to room temperature before being used.
• Oxalate or heparin plasma can also be used.
•
•
•
Do not use citrated plasma because citrate-utilizing bacteria E.g. Enterococci, Pseudomonas and Serratia may cause clotting of the
plasma (in tube test).
Occasionally, human plasma may contain inhibition substances which can interfere with coagulase testing.
It is therefore essential to test the plasma using a known coagulase positive S. aureus.
Slide test method (detects bound coagulase)
1. Place a drop of distilled water on each end of a slide or on two separate
slides
2.
Emulsify a colony of the test organism (previously checked by Gram staining) in each of the drops to make two thick suspensions.
➢Note: Colonies from a mannitol salt agar culture are not suitable for coagulase testing. The organism must first be cultured on
nutrient agar or blood agar.
3. Add a loopful (not more) of plasma to one of the suspensions, and mix
gently.
7. 7
2. Coagulase Test…Cont’d
4. Look for clumping of the organisms within 10 seconds.
5. No plasma is added to the second suspension. This is used to differentiate any granular appearance of the organism from true
coagulase clumping
Results
• Clumping within 10 seconds…...S. aureus
• No clumping within 10 seconds …No bound coagulase
Control
• Positive coagulase control …..S. aureus
• Negative coagulase control … E. coli or S. epidermidis
8. 8
2. Coagulase Test…Cont’d
Test tube method (detects free
coagulase) 1.
Take three small test tubes and label
as:
▪ T = test organism(18 – 24 hours broth culture)
▪
▪
2.
4.
5.
Pos = positive control (18 – 24 hours S. aureus broth culture)
Neg = Negative control (sterile broth)
* Nutrient broth is suitable. Do not use glucose broth.
Pipette 0.2ml of plasma in to each tube 3. Add 0.8ml of the test broth culture to tube T.
Add 0.8ml of the S. aureus culture to the tube labeled as ‘Pos’
Add 0.8ml of sterile broth to the tube labeled as ‘Neg’ 6. After mixing gently, incubate the three tubes at 35 – 37
o
C.
7. Examine for clotting after 1 hour.
– If no clotting has occurred, examine after 3 hours.
– If the test is still negative, leave the tube at room temperature over night and examine again by tilting the tube gently.
2. Coagulase Test…Cont’d
9. Results
• Clotting in the tube……….S. aureus
• No clotting ……………… Negative test
Note: There should be no clotting in the negative control tube.
3. Oxidase test/Cytochrome oxidase test
•The oxidase test is used to detect bacteria that produce the enzyme cytochrome oxidase which catalyze oxidation of reduced
cytochrome by oxygen molecule.
9
10. 10
• It assist in the identification of Pseudomonas, Neisseria, Vibrio, Brucella, and pasteurella species, which are oxidase positive.
3. Oxidase test/Cytochrome oxidase test…Cont’d
Principle
• A piece of filer paper is soaked with a few drops of oxidase reagent.
•
•
•
•
A colony of the test organism is then smeared on the filter paper. – Alternatively an oxidase regent strip can be used.
When the organism is oxidase-producing, the Tetramethyl-pphenylenediamine dihydrochloride in the reagent will be oxidized to
a deep purple colour.
Occasionally the test is performed by flooding the culture plate with oxidase reagent but this technique is not recommended for
routine use because the reagent rapidly kills bacteria.
It can however be useful when attempting to isolate N. gonorrhoeae colonies from mixed cultures in the absence of a selective
medium.
– The oxidase positive colonies must be removed and subcultured within 30 seconds of flooding the plate.
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3. Oxidase test/Cytochrome oxidase test…Cont’d
Material Required
• Fresh Oxidase reagent (Tetramethyle-p-phenylenediamine dihydrochloride, 1%)
•
•
•
Filter paper or oxidase regent strip Note:
Fresh oxidase reagent is easily oxidized.
When oxidized it appears blue and must not be used.
Method
1. Place a piece of filter paper in a clean petri
dish
2.
3.
4.
Add 2 or 3 drops of freshly prepared oxidase reagent,
Using a piece of stick or glass rod (not an oxidized wire loop), remove a colony of the test organism and smear it on the filter
paper.
Look for the development of a blue-purple colour within a few seconds as shown in the figure.
12. 3. Oxidase test/Cytochrome oxidase test…Cont’d
Result
• Blue – purple color …..positive Oxidase test (With in 10 sec)
• No blue – Purple color …Negative Oxidase test (With in 10 sec)
Note: Ignore any blue – purple color that develops after 10 seconds.
12
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3. Oxidase test/Cytochrome oxidase test…Cont’d
Method using an oxidase regent strip
1. Moisten the strip with a drop of sterile
water.
2.
3.
Using a piece of stick or glass rod (not an oxidized wire loop) remove a colony of the test organism and rub it on the strip.
Look for a red-purple colour within 10 seconds.
NB: Red-purple colour………….positive oxidase
test.
Controls
•Positive oxidase control: Pseudomonas aeruginosa
•Negative oxidase control: Escherichia coli
14. 14
4. Urease test
• This test is used to detect the enzyme urease, which breaks down urea into ammonia.
•
•
•
Testing for urease enzyme activity is important in differentiating enterobacteria.
Brucella and Proteus strains are strong urease producers.
Y. enterocolitica also shows urease activity (weakly at 35-37 o
C).
Principle
• The test organism is cultured in a medium which contains urea and the indicator phenol red.
•
•
•
When the strain is urease-producing, the enzyme will break down the urea (by hydrolysis) to give ammonia and carbon dioxide.
With the release of ammonia, the medium becomes alkaline as shown by a change in colour of the indicator to pink-red.
Ways of performing a urease test
–Using modified christensen’s urea broth.
–Using a Rosco urease identification tablet.
4. Urease test…Cont’d
15. 15
Method
A. Urease test using Christensen’s (modified) urea broth
• Inoculate heavily the test organism in a bijou bottle containing 3 ml sterile Christensen’s modified urea broth
•
•
Incubate at 35-37 o
C for 3-12 hours (preferably in a water bath for a quicker result).
Look for a pink colour in the medium.
Results
• Pink colour…………………..Positive urease test
• No pink colour……………… Negative urease test
4. Urease test…Cont’d
16. Fig. Urease test: The tube on the left is a positive reaction; the tube in the middle is a negative reaction and the tube on the
right is an un-inoculated control.
4. Urease test…Cont’d
B. Urease test using a Rosco urease tablet
•Urease identification tablets are available commercially
16
17. 17
• Prepare a dense ‘milky’ suspension of the test organism in 0.25 ml physiological saline in a small tube.
•
•
Add a urease tablet, close the tube and incubate at 35-37 o
C (preferably in a water bath for a quicker result) for up to 4 hours or
overnight.
Proteus and Brucella organism give a positive reaction within 4 hours.
Results
• Red/purple colour…………….positive urease test
• Yellow/orange……………….. Negative urease test
Control
• Positive urease control:
•
Proteus spp
Negative urease control: Salmonellae spp
5. Indole test
• The test detect the ability of an organism to produce indole from Tryptophan.
•
•
Testing for indole production is important in the identification of enterobacteria.
Most strains of E. coli, P. vulgaris, P. rettgeri,
Providencia species are indole positive organisms
and
18. 18
Principle
• The test organism is cultured in a medium which contains tryptophan.
•
•
Indole production is detected by Kovac’s or Ehrlich’s reagent which contains para-dimethylamino-benzaldehyde.
This reacts with the indole to produce a red coloured compound.
5. Indole test …Cont’d
Material required
• Kovac’s or Ehrlich’s reagent
• Bijou bottle/test tube
Method
Indole test using tryptone water and Kovac’s reagent.
1. Inoculate the test organism in a bijou bottle containing 3 ml of sterile tryptone
water.
2.
3.
4.
5.
Incubate at 35 – 37 o
C for up to 24 hours
Test for indole by adding 0.5ml of Kovac’s reagent and shake gently.
Examine for a red color in the surface layer with in 10 minutes.
Indole test …Cont’d
19. Results
• Red surface layer…………………Positive indole test
• No red surface layer………………. Negative indole test
Control
• Positive control ….. Escherichia coli
• Negative control…. Klebsiella pneumoniae
Fig. Indole test: The tube on the left with the red ring is positive for
indole production while the tube on the right shows a negative result.
6. Citrate utilization test
▪ The test detect the ability of an organism to use citrate as its only source of carbon.
▪ This test is one of several techniques used to assist in the identification of enteric bacteria.
Principle
•Some bacteria can obtain energy in a manner other than by the fermentation of carbohydrate by using citrate as source of carbon.
19
20. 20
• The utilization of citrate by a test bacterium is detected in citrate medium by the production of alkaline by-products.
•
•
•
The medium includes sodium citrate as the sole source of carbon and ammonium phosphate as the sole source of nitrogen.
Bacteria that can use citrate can also extract nitrogen from the ammonium salt, with the production of ammonia (NH+
), leading to
alkalinization of the medium.
In the presence of the indicator Bromothymol blue the medium will be converted from green (at pH 6.0) to blue (at a pH above
7.6).
Citrate utilization using Simmon’s citrate agar
Material required
• Simmon’s citrate medium/agar
• Inoculating loop
Method
1. Prepare slopes of the medium in bijou bottles as recommended by the manufacturer (store at 2-8
o
C
2.
3.
Using a sterile inoculation loop, streak the slope with a saline suspension of the test organism. Incubate at 35 o
C for 48 hours
Look for a bright blue colour in the medium
Results
• Bright blue-----------------------------------------Positive citrate test
• No change in colour of medium------------Negative citrate test
21. Controls
• Positive control ---------------------------- Klebsiella pneumoniae
• Negative control------------------------------------- Escherichia coli
Fig. Citrate utilization test: Left tube is a negative result. Right tube is a positive result.
7. Bile solubility test
21
22. 22
• This helps to differentiate S. pneumoniae, which is soluble in bile and bile salts, from other alpha-haemolytic streptococci (viridans
streptococci) which are insoluble.
Principle
• A heavy inoculum of the test organism is emulsified in physiological saline and the bile salt sodium deoxycholate is added.
•
•
This dissolves S. pneumoniae as shown by a clearing of the turbidity within 10-15 minutes.
Viridans and other streptococci are not dissolved and therefore there is no clearing of the turbidity.
23. 23
7. Bile solubility test…Cont’d
Material Required
•Sodium deoxycholate, 100 g/l (10% w/v)
•Physiological saline (sodium chloride, 8.5 g/l)
Method
•Although the bile solubility test can be performed by testing colonies directly on a culture plate or on a slide, a tube technique is
recommended because the results are easier to read.
Tube method
1. Emulsify several colonies of the test organism in a tube containing 2 ml sterile physiological saline, to give a turbid
suspension.
2.
3.
4.
Divide the organism suspension between two tubes
To one tube, add 2 drops of the sodium deoxycholate reagent and mix
To the other tube (negative control), add 2 drops of sterile distilled water and mix
24. 24
7. Bile solubility test…Cont’d
5. Leave both tubes for 10-15 minutes at 35-37 o
C.
6. Look for a clearing of turbidity in the tube containing the sodium deoxycholate.
Results
• Clearing of turbidity ………………probable S. pneumoniae
•
•
No clearing of turbidity…………… probable not S. pneumoniae
There should be no clearing of turbidity in the negative control tube to which distilled water was added.
Controls
• Bile solubility positive control:…. Streptococcus pneumonia
• Bile solubility negative control:… Enterococcus faecalis
25. 25
8. DNase test
• This test is used to identify S. aureus which produces deoxyribonuclease (DNase) enzymes.
• The DNase test is particularly useful when plasma is not available to perform coagulase test or when the results of a coagulase test are
difficult to interpret.
Principle
• Deoxyribonuclease hydrolyzes deoxyribonucleic acid (DNA).
•
•
•
•
The test organism is cultured on a medium which contains DNA.
After overnight incubation, the colonies are tested for DNase production by flooding the plate with a weak hydrochloric acid
solution.
The acid precipitates unhydrolyzed DNA.
DNase-producing colonies are therefore surrounded by clear areas due to DNA hydrolysis.
Material Required
• DNase agare plate
– Up to six organisms may be tested on the same plate.
•Hydrochloric acid, 1 mol/L (1 N)
26. 26
8. DNase test…Cont’d
Method
1. Divide a DNase plate into the required number of strips by marking the underside of the
plate.
2.
3.
4.
5.
6.
7.
8.
Using a sterile loop or swab, spot-inoculate the test and control organisms.
Make sure each test area is labelled clearly.
Incubate the plate at 35-37 o
C overnight.
Cover the surface of the plate with 1 mol/L hydrochloric acid solution.
Tip off the excess acid.
Look for clearing around the colonies within 5 minutes of adding the acid.
DNase test…Cont’d
27. Results
• Clearing around the colonies………………….DNase positive strain
• No clearing around the colonies……………… DNase negative strain
Controls
• Controls Postive DNase control:……..Staphylococcus aureus
• Negative DNase control: ……………Staphylococcus epidermidis
9. Litmus milk decolorization test
• This test is a rapid inexpensive technique to assist in the identification of Enterococci.
27
28. 28
• It is based on the ability of most strains of Enterococcus species to reduce litmus milk by enzyme action as shown by decolorization of
the litmus.
Note:
• Enterococci can also be identified using an aesculin hydrolysis test
Principle
• A heavy inoculum of the test organism is incubated for up to 4 hours in a tube containing litmus milk.
• Reduction of the litmus milk is indicated by a change in colour of the medium from mauve to white or pale yellow.
9. Litmus milk decolorization test…Cont’d
Material required
•Litmus milk medium
Method
• Using a sterile loop, inoculate 0.5 ml of sterile litmus milk medium with the test organism.
– Important: A heavy inoculum of the test organism must be used.
• Incubate at 35-37 o
C for up to 4 hours.
• Examining at half hour intervals for a reduction reaction as shown by a change in colour from mauve to white or pale yellow
– compare with the positive control.
Results
• White or pale yellow-pink colour……………….Suggestive of Enterococcus
29. 29
• No change or pink colour………………………. Probably not Enterococcus
Controls
• Positive control:
•
Enterococcus species
Negative control: Viridans Streptococci
10. Aesculin hydrolysis test
• This test can be economically performed using a Rosco bile aesculin tablet
➢ The test can be performed by placing a tablet on a blood agar plate inoculated with the test organism and incubating it at 35-37 o
C
overnight.
➢ A positive test is indicated by the tablet and colonies around it turning black/grey.
➢
▪
A negative test is shown by the tablet remaining white and no change in colour of the colonies.
Altematively, the test can be performed by making a dense suspension of the test organism in 0.25 ml of physiological saline in a
small tube, adding a tablet, and incubating at 35-37 o
C for 4 hours (or overnight).
▪ A positive reaction is shown by a black/grey colour in the medium.
11. MR-VP (methyl red-Vogues Proskauer) test
30. 30
• This test is used to determine
– The MR portion (methyl red) is used to determine if glucose can be converted to acidic products like lactate, acetate,
and formate.
– The VP portion is used to determine if glucose can be converted to acetoin (neutral end product).
• These tests are performed by inoculating a single tube of MRVP media with a transfer loop and then allowing the culture to
grow for 3-5 days.
•
•
After the culture is grown, about half of the culture is transferred to a clean tube.
One tube of culture will be used to conduct the MR test, the second tube serves as the VP test.
31. 31
11. MRVP test…Cont’d
A.
•
Methyl red (MR) test:
Methyl red is added to the MR tube.
➢ A red color indicates a positive result
– glucose can be converted into acidic end products such as lactate, acetate, and formate.
➢ A yellow color indicates a negative result,
– glucose is converted into neutral end products.
32. Figure: methyl red test
B. VP (Vogues Proskauer) test:
•First alpha-naphthol (also called Barritt’s reagent A) and then potassium hydroxide (also
called Barritt’s reagent B) are added to the VP tube.
32
33. 33
11. MRVP test…Cont’d
• The culture should be allowed for about 15 minutes for color development to occur.
•
If acetoin is produced then the culture turns to red color
(positive result).
• If acetoin is not produced then the culture appears yellowish in color (a negative result).
35. 35
11. MRVP test…Cont’d
• VP (Vogues Proskauer) test positive bacteria include
–Klebsilla spp, Enterobacter, spp and Serratia spp, Vibrio,
Staphylococci
Methyl Red positive bacteria include
–Escherchia coli, Citobacter spp., Salmonella spp, Proteus spp.,
Yersinia spp., Staphylococci
•MR-VP test positive bacteria include – Staphylococci
12. Triple sugar Iron (TSI) & Hydrogen sulfide production
(H2S)
•
• Looks at fermentation of glucose, lactose, and sucrose and checks if hydrogen sulfide and gas is produced in the process.
•
•
•
•
•
•
Basically a pH indicator will change the color of the media in response to fermentation.
The color change that occurs in the tube will indicate what sugar or sugars were fermented.
The presence of a black color indicates that H2S was produced.
In this media, H2S reacts with the ferrous sulfate in the media to make ferrous sulfide, which is black in colour.
To inoculate, use a needle to stab agar and then use a loop to streak the top slanted region.
In addition to TSI media, KIA media can be used to determine if H2S production.
38. 38
12. TSI…Cont’d
From left to right:
A.Uninoculated control
B.Red slant and red butt, no black color= no fermentation of
glucose, sucrose or lactose. No Hydrogen sulfide produced
C. Red slant and black butt= no lactose or sucrose fermentation, H2S has been produced
D. Red slant with yellow butt= no lactose or sucrose fermentation, Glucose is fermented, no H2S has been produced
E. Yellow slant, yellow butt and black coloration= Lactose, sucrose and glucose fermented, and H2S has been produced
F.Yellow slant, yellow butt and lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H2S has not been
produced but gas has been produced
G. Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented,
H2S has not been produced nor has gas been produced
13. Kligler Iron Agar (KIA)
• KIA is a composite medium containing glucose, lactose, phenol red and ferric citrate.
•
•
A yellow base indicates glucose fermentation.
A yellow base and slope indicates both glucose and lactose fermentation.
39. 39
• Bubble in the medium indicate gas production from glucose.
•
Blackening of the medium indicate H2S production.
14. Nitrate reduction test
• Nitrate broth is used for the test
Principle
•The test detects the ability of the organism to produce the enzyme nitrate reductase which reduces nitrate to nitrite.
Method
•
•Add 0.1 ml of the test reagent to the culture
•
Growing the bacteria for 4-5 days at 37 o
C in a broth containing 1% KNO3 .
The test reagent consists of a mixture of equal volume of solutions of Sulphonilic acid and a-naphthylamine in 5 N acetic acid.
Mixed just before use.
Result
• Positive ---------Red colour developing within a few minutes
• Negative--------No colour change
15. Additional non - biochemical test (Motility test)
40. 40
Motility test
•The motility test is not a biochemical test since we are not looking at metabolic properties of the bacteria.
• Rather, this test can be used to check for the ability of bacteria to migrate away from a line of inoculation.
• To perform this test, the bacterial sample is inoculated into motility media using inoculating straight wire.
• Simply stab the media in as straight a line as possible and withdraw the needle very carefully to avoid destroying the straight line.
•
•
•
•
After incubating the sample for 24-48 hours, observations can be made.
Check to see if the bacteria have migrated away from the original line of inoculation.
If migration away from the line of inoculation is evident then you can conclude that the test organism is motile (positive test).
Lack of migration away from the line of inoculation indicates a lack of motility (negative test result).
41. Fig. Motility test: Left tube is the result for Figure: Motility agar is a differential medium (Triphenyla non-motile bacterium. Right tube
is the tetrazolium chloride) .The organisms in the two tubes pictured result for a motile organism. on the right are motile.
16. Motility-Indole-Urea (MIU)
• MIU is a composie medium containing tryptone, phenol red, urea and a paper strip moistened in Kovac’s reagent.
41
42. 42
• It is inoculated by straight wire through the center of the medium
•
•
•
Non-motile organism grow only in the line of the inoculum, but motile organism grow through out the medium which become
turbid
Urease positive organism turn the medium red
Indole positive organism turn the Kovac’s strips red
17. Fermentation of carbohydrates
• Carbohydrates are complex chemical substrates that serve as energy sources when broken down by bacteria and other cells.
• Facultative anaerobic and anaerobic bacteria are capable of fermentation, an anaerobic process during which carbohydrates are
broken down for energy production.
• We can detect whether a specific carbohydrate has been fermented by looking for common end products of fermentation.
•
➢
➢
When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced:
Acid end products, or
Acid and gas end products.
Fermentation of carbohydrates…….
•In order to test for these fermentation products, inoculate and incubate tubes of media containing a single carbohydrate (such as
lactose or maltose), a pH indicator (such as phenol red) and a Durham tube (a small inverted tube to detect gas production).
43. • If the particular carbohydrate is fermented by the bacterium, acid end products will be produced which lowers the pH,
causing the pH indicator to change color (phenol red turns yellow).
▪ If gas is produced along with the acid, it collects in the Durham tube as a gas bubble (Fig.).
▪ If the carbohydrate is not fermented, no acid or gas will be produced and the phenol red will remain red.
43
46. 46
• Streptococcus pyogenes produces the enzyme pyrrolidonyl amino peptidase which is able to break down the substrate
L- pyrrolidonyl – beta – naphthylamide to produce beta-naphthylamine.
• The beta-naphthylamine can be detected by using N,N-D-dimethyl amino cinnamaldehyde reagent indicated by the
production of pink or cherry red color.
• Besides S. pyogenes, Enterococcus sp. and occasionally Streptococci belonging to groups C and G are also PYR positive.
•
•
Moisten the PYR disc slightly with distilled or deionized water. Do not
saturate.
Using a sterile loop, pick 2–3 well isolated, 18–24 h old colonies and rub into a small area of the PYR disc so that here is a
visible paste.
• After the test organism has been inoculated onto the disc, allow it to react for 2 min.
• After the incubation period, add one drop of PYR reagent.
19. ONPG (O-nitrophenyl galactosidase) test
•Lactose is fermented only when beta - galactosidase and permease are present. Deficiency of the later gives late fermentation.
True non-lactose fermenters do not possess Beta galactosidase.
•If beta galactosidase is present, a yellow colour is formed as the result of o-nitrophenol liberation.
•Place an ONPG disc in 0.1 ml 0.85%
sodium (physiological saline) in which emulsify a colony of the test organism
•Incubate at 35±2 oC for 6 h.
•If Beta galactosidase is present, a yellow colour is formed as the result of o-nitrophenol liberation.
•If there is no colour, incubate overnight to detect lactose fermenters.
chloride
47. 47
20. Lysine decarboxylase (LDC) test
✓This test will be useful for the identification and differentiation of
salmonellae (positive) and shigellae (negative).
✓Lysine is an amino acid that can be broken down by decarboxylase
enzymes possessed by some bacteria.
✓During this process, the carboxyl (COOH) group on the amino acid
molecule is removed, leaving alkaline end products that change the
color of the pH indicator.
✓The reactions work best when air is excluded (anaerobic conditions) from
the medium; therefore, the broth is layered
with mineral oil after inoculation and before incubation.
✓Obtain two tubes of Lysine Decarboxylase broth. ✓Inoculate one tube with
Shigella boydii and inoculate the other tube with E. coli. ✓Overlay each tube
with 2 ml of mineral oil. ✓Incubate at 35±2 °C for 48h.
✓*Check tubes for pretty purple color development for positive result, whereas
the negative result is indicated by yellow color
48. 21. Starch hydrolysis
• Amylase enzyme is
required
•
to hydrolyze the starch into dextrin and maltoses.
The final hydrolysis of this disaccharide catalyzed by
maltose, yields low molecular
weight, soluble
glucose molecules. The ability of the bacteria to
utilize starch can be performed by growing them on
starch agar medium. After the incubation, iodine in added to
culture plate to
or hydrolyzed.
detect whether the starch is utilized
• If the starch is hydrolyzed a clean zone around the
colony/growth of bacteria is observed. Instead, if the starch is
not utilized the iodine react with the starch and producing blue
colour
22. Satellitism test
• This test is useful to identify Haemophilus
influenza.
• Media used to grow H. influenzae must contain haemin or other
iron- containing porphyrin and nicotinamide adenine dinucleotide
(NAD)
• or its phosphate
(NADP).
48
49. 49
• The porphyrin requirement is referred to as growth factor X and the NAD or NADP requirement as growth factor
V.
•
•
Factor X is used by H. influenzae to produce essential respiratory enzymes such as cytochromes, catalase, and
peroxidase.
Factor V is used as an electron carrier in the organism’s oxidation – reduction
system.
22. Satellitism test….
Method
•Mix a loopful of Haemophilus growth in about 2 ml of sterile physiological saline or sterile peptone broth. Make sure none of the
chocolate agar medium is transferred.
•Using a sterile swab, inoculate the organism suspension on a plate of nutrient agar or trypticase soy agar, and a plate of blood
agar.
• Streak a pure culture of S. aureus across each of the inoculated
plates.
•
•
Incubate both plates at 35±2 o
C in the presence of CO2for 24h.
Examine the cultures for growth and satellite
colonies.
Interpretation
• H. influenzae shows growth on the blood agar plate but not on the nutrient agar plate, and the colonies near the column of S.
aureus growth are larger than those furthest from it. If satellite colonies are present on both plates the organism is probably of
Haemophilus species that requires only factor V, such as H. parainfluenzae. Blood contains X factor and inoculating
known S.aureus will provide V factor to the media which will help H. influenzae to grow. A rapid presumptive
identification of H. influenzae by satellitism can often be made if S. aureus is inoculated on two or three small areas of a
primary culture
50. 23.Protease activity
• A secreted bacterial protease may also act as an
exotoxin, and be an example of a virulence factor in
bacterial pathogenesis.
• Bacteria are inoculated on skim milk agar
plates.
•
•
It was incubated at 35°C for 12-24 h
If the clear zone is found around thebacterial
colonies/bacterial growth,
they are
of producing protease enzyme.
capable
50
51. 24. Lipase production test
• Lipases are expressed
secreted organisms during the
infection.
by
pathogenic
• Therefore,
lipolytic
activity, which
the persistence
may
and
contribute to
virulence of microorganisms
• Tributyrin agar was prepared and inoculated
with bacterial culture and incubated at 37°C for overnight.
• A clear zone around the colonies observed is positive for
lipase production.
51
52. 25. Biofilm production test
▪ The extracellular slime (biofilm) produced by bacteria
has been shown to interfere with several human neutrophil
functions, such as chemotaxis, degranulation,
phagocytosis and antibiotic resistant.
▪Agar plates with Congo red stain are inoculated with
different bacterial strains and incubated aerobically at 37°C
for 24 h.
▪Bacteria which produced black colonies with dry
crystalline consistency are slime positive, whereas those
showing pink colonies are slime negative.
crystalline consistency.
26. Beta-lactamase test
•Production of beta-lactamase enzymes that destroy the
beta- lactam ring of penicillin and cephalosporin
(commonest form of resistance). The most reliable
way to detect beta- lactamase-producing strains of N.
gonorrhoeae is to use the nitrocefin test.
•Use sterile forceps to place a nitrocefin disc on a clean
slide.
52
▪ Positive for slime production by the
formation of black colonies
with
dry
53. 53
• Add a drop of distilled water to the disc and allow it to absorb so that the disc is moistened, but not wet. Do not oversaturate
the discs.
• Touch a sterile swab or loop to a characteristic colony from fresh (18 – 24 h old) pure culture plate.
•
•
•
Rub the colony on the moistened disc so that the growth goes into the filter paper disc.
Examine the disc for 5 min.
If the reaction is positive, the areas of the disc containing growth will turn a characteristic red/pink color. Reactions typically
occur within 5 min.
• Strains for which the inoculum on the nitrocefin disc turns red/ pink are considered beta-lactamase positive. Strains for
which the inoculum on the nitrocefin disc does not change color are considered beta-lactamase negative.
