Biochemical Tests for Identification

of Bacteria
 Biochemical tests :
These tests are performed for identification of bacteria. 

All of these tests depend upon production of certain enzymes by 

the bacteria.

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.
This enzyme converts hydrogen peroxide into water and 
oxygen.
 Principle:

Catalase producing bacteria will produce O2 when mixed with H2O2.

2 H2O2 -------------> 2 H2O and

O2 (Bubbles)
Methods:

1-Tube Test .1

2-Slide Test .2

Slide Method: Take a drop of 3%

H2O2 on a glass slide .

Mix it with a small quantity of test bacteria.

Results:
Active bubbling . . . . . . . . . . . . Positive catalase test. 
No bubbles . . . . . . . . . . . . . . Negative catalase test. 
Caution: Performing the test on a slide is not recommended 
because of the risk of contamination from active
bubbling.

When the rapid slide technique is used, the hydrogen peroxide

solution should be added to the organism suspension after
placing the slide in a petridish. The dish should then be
covered immediately, and the preparation observed for
bubbling through the lid.
 Tube Method
Pour 2–3 ml of the hydrogen peroxide solution into a test 
tube.
Using a sterile wooden stick or a glass rod (not a nichrome wire loop), 
remove several colonies of the test organism and immerse in the hydrogen
peroxide solution.
Important: Care must be taken when testing an organism cultured on a 
medium containing blood because catalase is present in red cells & a false
positive reaction may occur.
Look for immediate bubbling. 
Results
Active bubbling . . . . . . . . . . . . Positive catalase test. •
No bubbles . . . . . . . . . . . . . . Negative catalase test. •
 - Coagulase Test:
This test is used to identify Staph. aureus, which produces coagulase enzyme.
Principle: Coagulase
causes plasma to clot by converting fibrinogen to fibrin. Two types of coagulase
are produced by most strains of Staph. aureus:

Free coagulase 

which converts fibrinogen to fibrin by activating a coagulase-reacting factor 

present in plasma. Free coagulase is detected by clotting in the tube
test.
Bound coagulase(clumping factor) 
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.
A tube test must always be performed when the result of a slide test is not clear, or 
when the slide test is negative and Staphylococcus has been isolated from a
serious infection.
Requirements :
Anticoagulated human plasma or rabbit plasma ( by EDTA, 
oxalate or heparin).
The plasma should be allowed to warm to room temperature 
before being used.
Do not use citrated plasma because citrate-utilizing bacteria e.g. enterococci & 
Pseudomonas may cause clotting of the plasma (in tube
test).
1- Slide test method (detects bound coagulase):
Place a drop of distilled water on each end of a slide or on two 
separate slides.
Emulsify a colony of the test organism in each of the drops to make 
two thick suspensions.
Add a loopful (not more) of plasma to one of the suspensions, and mix 
gently.
Look for clumping of 
the organisms within 10 seconds.
Results
Clumping within 10 sec 
. . . . . . Staph. aureus.
No clumping within 10 sec 
. . . . . No bound Coagulase.
2- Tube test method (detects free coagulase):
Label two small test tubes one as: Test organism (18–24 h broth culture) 
and the other as: Control (sterile broth).
Pipette 0.2 ml of plasma into each tube. 
Add 0.8 ml of the test broth culture to “Test” tube . 
Add 0.8 ml of sterile broth to “Control” 
After mixing gently, incubate the tubes at 35–370C for 6-12 hours and 
examine hourly.
If the test is still negative, leave the tube at room temperature overnight and 
examine again.
Note: When looking for clotting, tilt each tube gently.
Results
Clotting of tube contents . . . . . . . . . . . Staph. aureus. 
No clotting ………………. Not Staph. aureus. 
 - DNA-ase test
This test is used to help in the identification of Staph. aureus which produces 
deoxyribonuclease (DNAase) enzymes.

The DNA-ase test is particularly useful when plasma is not available to 

perform a 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 DNA-ase 
production by flooding the plate with a weak hydrochloric acid
solution.

The acid precipitates unhydrolyzed DNA. 

DNA-ase-producing colonies are therefore surrounded by clear areas 
due to DNA hydrolysis.
Requirements
DNA-ase agar. 
Hydrochloric acid solution 1 mol/l (1N). 

Method
Using a sterile loop or swab, inoculate the test and control 
organisms.
Incubate the plate at 35–37 0C overnight. 
Cover the surface of the plate with 1 mol/l hydrochloric acid 
solution.
Look for clearing around the colonies within 5 minutes of adding the 
acid.
Results
Clearing around the colonies . . . . . . . . . . 
DNA-ase positive strain Staphylococcus
aureus.

No clearing ………… 

Negative DNA-ase Staphylococcus 

epidermidis.
Uses: use in identification of microorganisms.
Classification:
1. Test for metabolism of carbohydrates: sugars
fermentation test and O/F test.
2. Test for specific break down of product: MR &
VP.
3. Test to show ability to utilize a specific
substrate: citrate utilization test.
4. Test for metabolism of proteins and amino
acids: H2S production test indole production
test
5. Test for enzymes: catalase, oxidase & urease.
1. Indole production test:
Aim: This test assist in the identification of gram 
negative rods).
Principle: The organism break down the treptophan
and produce Indole which can be detected by
Kovac,s reagent (4 (p) – dimethylaminobenzaldehyde).
Requirments: * Peptone water
* kovac,s reagent
Method:
 Using sterile loop inoculate the tested
organism into 2 ml peptone water or tryptone
water.
 Incubate the tube at 37oC for 24 hrs.

 Add 0.5 ml of kovac,s reagent , shake gently and 
examine for red colour ring within 10 mins.

Results: 

Red surface layer   ………….+ve test.

No red surface layer ……..…‐ve test.

Positive result : …………..… E. coli.

Negative result………………Klebsiela.
2. Citrate utilization test

Aim: This test assist in the identification of gram

negative rods
Principle: It is depend on the ability of the organism
to use citrate as its only source of carbon and
ammonia as its only source of nitrogen. If it can not
use citrate and ammonia then it will not grow. If it
can use citrate and ammonia, then the bacteria will
grow and the media will turn a bright blue as a
result of an increase in the pH of the media.
Requirements: * Koser’s Citrate broth
* or Simmo’n citrate agar
Method:
 Using sterile straight wire, inoculates the test
organism into 2 ml Koser’s citrate medium.
 Incubate the medium at 37oC for 24 hrs.
 Look for color change and/or turbidity on the
medium.
Results: 
Blue color and/or turbidity in the medium…+ve test.
No change in the medium……………………... ‐ve test.

Positive result : …… Klebsiela.
Negative result..………E.coli.
3. Urease production test

Aim: This test assist in the identification of gram

negative rods
Principle: The enzyme urease hydrolyzes urea,
producing ammonia (alkaline pH), which changes the
colour of phenol red indicator to red‐pink colour.
Requirement: * Christensen's urea medium.
Method:
 Using sterile straight wire inoculates the test
organism into slope surface of Christensen's urea
medium.
 Incubate the medium at 37oC for 24 hrs.
 Look for color change on the medium.
Results: 

Pink color on the medium………+ve test.

No pink color on the medium…… ‐ve test.

Positive result : ………………… Klebsiela.

Negative result……………………E.coli.
4. Oxidase test
Aim: This test assist in the identification of gram
negative rods
Principle: The enzyme oxidase oxidize
phenylenediamine to deep purple colour.
Requirement: * oxidase reagent (Tetramethyl‐p‐
phenylenediamine dihydrochloride).
Method:
 Place apiece of filter paper in a clean petri‐dish
and add 2 – 3 drops of freshly prepared oxidase
reagent.
 Using a piece of wooden stick or glass (not an
oxidized wire loop) remove a colony of the test
organism and smear it on the filter paper.
 Look for color change within few seconds.

Results: 
blue purple within 1o second…………+ve test.
No change within 1o second ……….. ‐ve test.
Positive result: vibrio, Pseudomonas & Neisseria
Negative result………Salmonella & Shigella

* Acidity inhibit oxidase result …….?
5. Kligler Iron Agar (KIA):
Aim: This test assist in the identification of gram negative
rods.
Principle: based on fermentation of glucose and lactose, Gas
production, and hydrogen sulfide is production.
Fermentation of sugar(s) change a pH , so indicator (phenol 
red) will change the colour of the media from red to yellow. 
The presence of a black color indicates that H2S was 
produced. H2S reacts with the ferrous sulfate in the media to 
make ferrous sulfide, which is black. 
Requirement: * KIA.
Method:
 Inoculate, using a straight wire loop  to stab agar 
butt, close to the opening and then streak the top 
slope (zigzag). 
 Incubate the medium at 37oC for 24 hrs.
 Look for color change on the medium.
Results:
Glucose  Lactose  H2S  Gas 
fermentation fermentation production production

Yellow butt Yellow slope Black tube  Cracks and 

end bubbles
6. MR VP (Methyl Red‐Vogues Proskauer) test:

Aim: This test assist in the identification of gram – ve rods).

Principle: The MR test is used to determine if glucose can be
converted to acidic products (lactate, acetate, and formate).
The VP test is used to determine if glucose can be converted
to acetoin.
Requirments: *2 tubes glucose 6 phosphate peptone water
* Methyl red indicator.
* 40% KOH.
* 5% alpha naphthol.
•Method:
• Using sterile loop inoculate the tested organism
into 2 ml glucose 6 phosphate peptone water.
• Incubate at 37oC for 24 hrs.
A. MR test:
• Add methyl red.
• Look for colour change.
B. VP test:
• add alpha‐naphthol 
• add 40% KOH
• look for colour development within 15 minutes. 
Results: 
A. MR test:
Red colour………………………………+ve test.
Yellow colour ……………………..….. ‐ve test.
B. VP test
Red colour within 15 minutes ……+ve test.
Yellow to cooper colour within 15 minutes .. ‐ve test.

E.coli:  MR (+ve) VP (‐ve)
Klebsiela MR (‐ve) VP (+ve)
1 MR (-ve)
2 MR (+ve)
7. Motility test (Non‐Biochemical Tests):

Aim: This test assist in the identification of gram

negative rods.

Principle: This test use to check the ability of bacteria

to migrate away from a line of inoculation via flagella.

Requirement: * semi solid media (motility media )

Method:

1. Inoculate into motility media using straight wire

loop, stab the media in as straight a line as possible
And withdraw the loop very carefully to avoid
destroying the straight line.
2. Incubate at 37oC for 24 hrs.
Results:
 Migration away from the original line of
inoculation motile organism.
 Lack of migration away from the line of
inoculation non motile organism.
motile Non
motile
8. Carbohydrate Utilization test:
Aim: This test assist in the identification of gram
negative rods.
Principle: Bacteria produce acidic products when they
ferment certain carbohydrates, this lower the pH, and
change indicator colour. If there is no fermentation
the media remains without change. If gas is produce
as a by product of fermentation, then the Durham
tube will have a bubble in it.
Requirement: * (Set of sugar )
Method:
• Using sterile loop inoculate the tested organism
in the media.
• Incubate at 37oC for 24 hrs.
Results: 
Look for colour change* (fermentation)
Look for bubble in the Durham tube (gas) 
* According to the indicator
9. Oxidation Fermentation test (OF test)
Aim: This test assist in the identification of gram negative
rods.

Principle: Oxidation: (utilization of carbohydrate in the

presence of oxygen). Fermentation (utilization of
carbohydrate in the presence or absence of oxygen.
Carbohydrates utilizaton produce acid, this lower the pH,
and change indicator colour.

Requirement: * 2 tubes Huge and Lephthon media

* Sterile wax.
Method:

 Inoculate, using a straight wire loop to stab agar

butt, close one tube by sterile wax.

 Incubate the medium at 37oC for 24 hrs.

 Look for color change on the medium.

Results:

Reaction Closed  Opened  Example

Tube Tube
Oxidation Green Yellow Pseudomonas sp.
Fermentation Yellow Yellow E. coli
No sugar  Green Blue Alkaligenes
utilization feacalis
Technical Yellow Green
error
10. Analytical Profile Index (API ) 
Biochemical Tests for Gram
Positive Bacteria
Catalase Test
• This test is used to differentiate between
catalase producing organisms,
(Staphylococci) and non catalase producing
organisms, (Streptococci).

Principle

– Catalase acts as a catalyst in the breakdown of

hydrogen peroxide to oxygen and water.

– 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.
Catalase Test
Requirements

– Hydrogen peroxide, 3%, sterile wooden stick or glass rod

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

Results

– Active bubbling ………………………….. Positive

– No bubbles ………………………………... Negative

Controls

– Positive ……………………………………. Staphylococcus spp

– Negative …………………………………... Streptococcus spp
Catalase Test

Negative Positive
Catalase Test
• Important

– The wire loop must not be used, as false positives

may occur

– Performing test on slide is not recommended,

because of the potential risk of aerosol production

– If a slide test has to be performed, then place slide

in a petri dish, and add hydrogen peroxide to the
test organism suspension, and immediately cover
the petri dish
Coagulase Test
This test is used to identify the Staphylococcus
aureus, which produces the enzyme coagulase

• Principle

– Coagulase causes plasma to clot by converting fibrinogen to

fibrin. Two types of coagulase are produced by most strains
of Staphylococcus aureus

• Free coagulase which converts fibrinogen to fibrin by activating a

coagulase reacting factor present in plasma. Free coagulase is
detected by clotting in the tube test

• Bound coagulase (clumping factor) 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
Coagulase Test
• Requirements

– EDTA anti-coagulated human plasma or rabbit plasma, slide, wire loop

• Slide test method

– Place a drop of distilled water on each end of a slide

– Emulsify a colony of the test organism in each of the drops

– Add a loopful of plasma to one of the suspensions, and mix gently

– Look for clumping of the organisms within 10 seconds

• Results

– Clumping within 10 seconds ……………….. Staphylococcus aureus

– No clumping within 10 seconds ………………. No bound coagulase

• Controls

– Positive coagulase control ………………………… Staphylococcus aureus

– Negative coagulase control ………………………. Staphylococcus epidermidis
Coagulase Test
• Tube Test Method

– Take 3 test tubes and label them as T, P, and N

– Pipette 0.2 mL of plasma into each tube

– Add 0.8 mL of the test broth culture to tube T

– Add 0.8 mL of the Staphylococcus aureus culture to the tube P

– Add 0.8 mL of the sterile broth to the tube N

– After mixing gently, incubate the three tubes at 37° C

– Examine for clotting after one hour

– If no clotting has occurred, examine after 3 hours. If still no clotting, then leave tubes
overnight

• Results

– Clotting of tube contents or clot in tube ………………….. Staphylococcus aureus

– No clotting …………………….………………………………..……. Negative test
Coagulase Test

Positive Negative
Coagulase Test
• Important

– A tube test must always be performed when the result of a slide test is not
clear, or when the slide test is negative and Staphylococcus has been
isolated from a serious infection

– The plasma used should be preferably pooled

– 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 inhibitory substances which can

interfere with test results. It is therefore essential to test the plasma using a
known coagulase positive Staphylococcus aureus

– Virulent strains of Yersinia pestis are also coagulase positive

DNase Test
• This test is used to identify Staphylococcus aureus which produces DNase
enzyme

• It is particularly useful when plasma is not available to perform a coagulase test

or when the results of a coagulase test are difficult to interpret

• Principle

– DNase hydrolyzes 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 HCL solution.

– The acid precipitates unhydrolyzed DNA.

– DNase producing colonies are therefore surrounded by clear areas due to DNA
hydrolysis.
DNase Test
• Requirement

– DNase agar plate, HCL 1N

• Method

– Divide a DNase plate into the required number of areas and label them

– Using a sterile loop or swab, spot inoculate the test and control organisms

– Incubate the plate at 37° C overnight

– Cover the surface of the plate with 1N HCl solution. Tip off the excess acid

– Look for clearing around the colonies within 5 minutes of adding the acid

• Results

– Clearing around the colonies ……. DNase positive strain

– No clearing around the colonies ….. DNase negative strain

• Controls

– Positive DNase control …….. Staphylococcus aureus

– Negative Dnase control …… Staphylococcus epidermidis
DNase Test
Bile Solubility Test
• This test helps to differentiate Streptococcus pneumoniae, which is soluble in
bile and bile salts, from other alpha hemolytic Streptococci (Viridans) 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 Streptococcus 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

• Requirement

– Sodium deoxycholate, 100 g/L

– Physiological saline
Bile Solubility Test
• Tube Method

– Emulsify several colonies of the test organism in a tube containing 2 mL sterile saline,
to give a turbid suspension

– 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

– Leave both tubes for 10-15 minutes at 35-37° C

– Look for a clearing of turbidity in the tube containing the sodium deoxycholate

• Results

– Clearing of turbidity …………………………… Probably S. pneumoniae

– No clearing of turbidity ………………………. Probably Not S. pneumoniae

• Controls

– Bile solubility positive control ………………….. S. pneumoniae

– Bile solubility negative control …………………. E. faecalis
Bile Solubility Test
Bile Solubility Test
• Important

– Bile solubility test can be performed by testing

colonies directly on a culture plate or on a slide

– The tube method is recommended because the

results are easy to read

– Some strains of S pneumoniae are not dissolved by

bile salts

– Occasionally some strains of viridans streptococci

give a positive test
Litmus Milk De-colorization Test
• This test is a rapid inexpensive technique to assist in the
identification of Enterococci

• It is based on the ability of most strains of Enterococcus spp to

reduce litmus milk by enzyme action which is shown by
decolorization of litmus

• 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 color of the

medium from mauve to white or pale yellow

• Requirements

– Litmus milk medium

Litmus Milk De-colorization Test
• Method

– Using a sterile loop, inoculate 0.5 mL of sterile litmus milk medium

with the test organism

– Incubate at 35-37° C for up to 4 hours, examining at half hour

intervals for a reduction reaction

– The reduction is shown by a color change from mauve to white or

pale yellow

• Results

– White or pale yellow-pink color …… Suggestive of Enterococcus

– No change or a pink color …….. Probably not Enterococcus

• Controls

– Positive control …….. Enterococcus spp

– Negative control …… Viridans streptococci
Culture Media
Types of Culture Media
Msc: Yousif Elfatih Yousif
Omdurman Islamic University
 Blood agar (B.A)

 Use : For the isolation, cultivation and

detection of hemolytic activity of streptococci,
pneumococci and other particular fastidious
microorganisms.
 Control organisms :
Streptococcus pneumoniae : Good growth,
Alpha - hemolysis Streptococcus
pyogenes : Good growth, Beta-
hemolysis
 Incubate: aerobic 35° C, 24 hrs.
Shelf life : 4 weeks
Blood Agar
α-Haemolysis on Blood
Agar
ß- Haemolysis on Blood
Agar
 Chocolate Agar

 Use : For the isolation and cultivation of a

variety of fastidious microorganism.
 Control organisms :
Hemophilus influenzae and Neisseria
gonorrhoeae : Good growth
 Incubate : CO2 ,35° C ,24 hrs.
Shelf life : 4 weeks
Chocolate Agar
 MacConkey agar

 Use : For the selective isolation, cultivation and differentiation of

coliforms and enteric pathogens based on the ability to ferment
lactose. Lactose–fermening organisms appear as red to pink
colonies. Lactose-nonfermenting organisms appear as colorless or
transparent colonies
 Control organisms :
Escherichia coli : Pink colonies (lactose fermentation)
Proteus mirabilis : Colorless colonies ,no spreading
Enterococcus sp. : No growth
 Incubate : Aerobic, 35° C, 24 hrs.
Shelf life : 4 weeks
MacConkey agar
Lactose Fermenting Colonies
on MacConkey Agar
Mueller-Hinton agar (MHA)

 Use : For antimicrobial susceptibility testing of

a variety of nonfastidious, rapidly-growing
microorganisms.
 Control organisms :
Escherichia coli ATCC25922 ,
Staphylococcus aureus ATCC 25923,
and Pseudomonas aeruginosa ATCC 27853 :
Growth ,Inhibition Zone followed Standard
interpretative
Incubate : Aerobic 35° C, 24 hrs 
Antimicrobial Sensitivity
Test by Disc Diffusion
Method
Salmonella-Shigella agar
(SS)

Use : For the selective isolation and differentiation of pathogenic 

enteric bacilli, especially those belonging to the genus Salmonella.
This media is not recommended for the primary isolation of
Shigella species. Lactose-fermenting bacteria such
as Escherichia coli or Klebsiella pneumoniae appear as small
pink or red colonies. Lactose-nonfermenting bacteria such as
Salmonella species, Proteus species and Shigella species appear
as colorless colonies. Production of H2S by Salmonella species
turns the center the colonies black.
Control organisms : 
Salmonella typhi : Colorless colonies, black
centers. Escherichia coli : Pink colonies
Incubate : Aerobic 35° C, 24 hrs 
Salmonella-Shigella agar
plate (SS)
Eosin Methylene Blue Agar
(EMB)

Use : For the isolation, cultivation and differentiation of Gram- 

negative enteric bacteria based on lactose fermentation. Bacteria
that ferment lactose, especially the coliform bacterium Escherichia
coli, Appear as colonies with green metallic sheen or blue–black to
brown color. Bacteria that do not ferment lactose appear as
colorless or transparent light purple colonies
Control organisms : 
Escherichia coli : Good growth, green metallic sheen.
Klebsiella pneumoniae : Good growth, purple colonies, no sheen.
Shigella flexneri : Good growth, transparent colonies (lactose
negative)
Incubate : Aerobic 35° C, 24 hrs 
Eosin Methylene Blue Agar
(EMB)
Thiosulfate Citrate Bile Salt
Sucrose agar (TCBS)

Use : For the selective isolation of Vibrio cholerae and 

Vibrio parahaemolyticus from a variety of clinical
specimens and in epidemiological investigations.
Control organisms : 
Vibrio cholerae : Growth (yellow colonies)
Vibrio parahaemolyticus : Growth (green colonies)
Staphylococcus aureus : No growth
Incubate : Aerobic 35° C, 24 hrs 
Thiosulfate Citrate Bile Salt
Sucrose agar plate (TCBS)
Triple Sugar Iron agar slant
(TSI slant)

Use : For the differentiation of members of the 

Enterobacteriaceae base on their fermentation of
lactose, sucrose and glucose and the production of
H2S.
Control organisms : 
Escherichia coli : A/A (G)
Shigella flexneri : K/A
Proteus vugaris : K/A, H2S
Pseudomonas aeruginosa : K/N
Incubate : Aerobic 35° C, 24 hrs 
Triple Sugar Iron agar slant
(TSI slant)
Motility Indole Lysine
Medium (MIL slant)

Use : For the cultivation and differentiation of members 

of the Enterobacteriaceae on the basis of motility,
lysine decarboxylase activity, lysine deaminase activity
and indole production.
Control organisms : 
Proteus vulgaris : Motile / Indole/ LDC/ LDM ; +/+/-
/+
Klebseilla pneumoniae : Motile / Indole/ LDC/ LDM ; -/-
/+/-
Incubate : Aerobic 35° C, 24 hrs 
Motility Indole Lysine
Medium (MIL slant)
Christensen Urea Agar slant

Use : For the differentiation of a variety of 

microorganisms, especially members of the
Enterobacteriaceae, aerobic actinomycetes,
streptococci and nonfermenting Gram-negative
bacteria, on the basis of urease production.
Control organisms : 
Proteus mirabilis : Pink throughout (positive)
Klebsiella pneumoniae : Pink slant (partial positive)
Escherichia coli : Yellow (negative)
Incubate : Aerobic 35° C, 24 hrs 
Christensen Urea Agar slant
Simmons’ Citrate Agar slant

Use : For the differentiation of Gram-negative bacteria 

on the basis of citrate utilization. Bacteria which can
utilize citrate as sole carbon source turn medium blue.
Control organisms : 
Klebsiella pneumoniae : Growth and blue color
(positive)
Escherichia coli : No growth, remains green
(negative)
Incubate : Aerobic 35° C, 24 hrs 
Simmons’ Citrate Agar slant
Bile Esculin Agar slant

Use : For the isolation and presumptive 

identification of enterococci (Group D
streptococci).
Control organisms : 
Enterococcus spp. : Good growth, black
Alpha-Hemolytic Streptococcus, not group D :
No growth, colorless on media
Incubate : Aerobic 35° C, 24 hrs 
Bile Esculin Agar slant
 Selenite-F Broth (SF)

Use : For the isolation and cultivation of 

Salmonella species from feces and other
specimens.
Control organisms : 
Salmonella spp. : Growth
Escherichai coli : No growth
Incubate : Aerobic, 35° C , 24 hrs 
Selenite-F Broth (SF)
Lowenstein-Jensen Medium
(L - J)
Use : For the cultivation and differentiation of Mycobacterium 
species. M.tuberculosis appears as granular ,rough, dry colonies.
M.kansasii appears as smooth to rough photochromogenic
colonies. M.gordonae appears as smooth yellow- orange
colonies. M.avium appears as smooth, colorless colonies.
M.smegmatis appears as wrinkled, creamy white colonies. Also
used for the cultivation and maintenance of Gordona
species, Nocardia species, Rhodococcus species, and
Tsukamurella paurometabolum.
Control organisms : 
Mycobacterium phlei : Good growth
Uninoculated slant : No growth
Incubate : Aerobic 35° C, 3 days 
Lowenstein-Jensen Medium
(L - J)
Stuart Transport medium

Use : For transport of swabs of clinical material 

containing upper respiratory tract, enteric
pathogens and other fastidious organisms.
Control organisms : 
Streptococcus pyogenes : Growth
Uninoculated medium : No growth
Incubate : Aerobic 35° C, 24 hrs 
Stuart Transport medium
• Culture medium or growth medium:
is a liquid or gel designed to support the 
growth of microorganisms
• The purpose of using cultural 
techniques:
in microbiology is to demonstrate the 
presence of organisms which may be 
causing disease, and when indicated, to 
test the susceptibility of pathogens to 
antimicrobial agents.
• Culture media can be classified;
I. Based on their consistency as:

1. Solid
2. Semi‐solid
3. Fluid
 Liquid medium

Solid medium Semisolid medium
II. Based on the constituents/ 
ingredients
1. Basic
2. Enriched
3. Enrichment
4. Selective
5. Indicator
6. Transport
7. Identification
III.Based on Oxygen requirement:

1. Aerobic media
2. Anaerobic media
1. Basic media:
These are simple media such as nutrient 
agar and nutrient broth that will support 
the growth of microorganisms that do not 
have special nutritional requirements. 
They are often used in the preparation of 
enriched media, to maintain stock 
cultures of control strains of bacteria, and 
for subculturing pathogens from 
differential or selective media prior to 
performing biochemical and serological 
identification tests.
Nutrient agar media
2. Enriched media:
Enriched media are 
required for the growth of 
organisms with exacting 
growth requirements such 
as H. influenzae, Neisseria 
species, and some 
Blood agar
Streptococcus species. 
Basic media may be enriched with whole or 
lyzed blood or serum.
3. Enrichment media:
This term is usually 
applied to fluid selective 
media which contain 
substances that inhibit 
the growth of unwanted 
organisms, e.g. Selenite F 
broth which is often used 
as an enrichment  Selenite F Broth

medium for Salmonella 
in faeces.
4. Selective media:
These are solid media which contain 
substances (e.g. bile salts or other 
chemicals, dyes, antibiotics) which inhibit 
the growth of one organism to allow the 
growth of another to be more clearly 
demonstrated. A selective medium is used 
when culturing a specimen from a site 
having a normal microbial flora to 
prevent unwanted contaminants 
overgrowing a pathogen.
Eg:
• Mac Conkey’s medium for gram negative 
bacteria
• TCBS – for V.cholerae
• LJ medium – M.tuberculosis
• Potassium tellurite medium – Diphtheria 
bacilli
• MSA medium _ for  S.aureus
Mac Conkey’s medium TCBS
Potassium Tellurite media LJ media
• MSA medium
5. Indicator (differential) media:

These media contain an indicator which 
changes its colour when a bacterium grows in 
them.
• On this media, dyes or other substances are 
added to differentiate microorganisms. Many 
differential media distinguish between 
bacteria by incorporating an indicator which 
changes colour when acid is produced 
following fermentation of a specific 
carbohydrate e.g.
_Blood agar
_Mac Conkey’s medium
_Christensen’s urea medium
• Lactose fermenters – Pink colonies
• Non lactose fermenters – colourless colonies
Urease medium
6. Transport media: 
These are mostly semisolid media that 
contain ingredients to prevent the 
overgrowth of commensals and ensure the 
survival of aerobic and anaerobic 
pathogens when specimens cannot be 
cultured immediately after collection.
Examples of transport media include:
_Cary‐Blair medium 
_ Amies transport medium 
7. Identification media:
These include media to which substrates 
or chemicals are added to help identify 
bacteria isolated on primary cultures. 
Examples include peptone water sugars, 
urea broth, and Kligler iron agar. 
Organisms are mainly identified by a 
change in the colour of the medium and 
or the production of gas. Organisms used 
to inoculate identification media must be 
first isolated in pure culture.
Anaerobic media
• These media are used to grow anaerobic organisms.
• Eg: Robertson’s cooked meat medium, Thioglycolate 
medium.
 Biological assay media: (specialized 
media )
• E.g: Muller‐Hinton medium   for antimicrobial 
sensitivity testing.
Culture methods include:
1. Streak culture
2. Lawn culture
3. Stroke culture
4. Stab culture
5. Pour plate method
6. Liquid culture
7. Anaerobic culture methods
STREAK CULTURE
• Used for the isolation of bacteria in pure culture from 
clinical specimens.
• Platinum wire or Nichrome wire is used.
• One loopful of  the specimen is transferred onto the 
surface of a well dried plate.
• Spread over a small area at the periphery.
• The inoculum is then distributed thinly over the plate 
by streaking it with a loop  in a series of parallel lines 
in different segments of the plate.
• On incubation, separated colonies are obtained over 
the last series of streaks.
Streak culture
LAWN CULTURE
• Provides a uniform surface growth of the bacterium.
• Uses
– Antibiotic sensitivity testing.
STROKE CULTURE
• Stroke culture is made in tubes 
containing agar slope / slant.
STAB CULTURE
• Prepared by puncturing a suitable medium –
gelatin or glucose agar with a long, straight, 
charged wire.
 Stab culture

Gelatin liquefaction Oxidation – Fermentation 

medium
LIQUID CULTURES
• Liquid cultures are inoculated by adding the inoculum
with pipettes or syringes.
• E.g.
– Blood culture
Blood culture bottles
 ANAEROBIC CULTURE METHODS
• Anaerobic bacteria differ in their requirement and 
sensitivity to oxygen.
• Cl.tetani is a strict anaerobe – grows at an oxygen 
tension < 2 mm Hg.
Methods:
_Displacement of oxygen with other gases
Eg: Candle jar
_Chemical method
Eg: Anaerobic jar
Candle jar for anaerobic culture
 Enterobacteriaceae (2)
Biochemical reactions
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University

Dr. Nabil El Aila

Diagnostic Microbiology
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Dr. Nabil El Aila

Diagnostic Microbiology
 Characters of Enterobacteriaceae
• All Enterobacteriaciae
– Gram-negative rods
– Ferment glucose with acid production
– Reduce nitrates into nitrites
– Oxidase negative
• Facultative anaerobic
• Motile except Shigella and Klebsiella
• Non-capsulated except Klebsiella
• Non-fastidious
• Grow on bile containing media (MacConkey agar)
Dr. Nabil El Aila
Diagnostic Microbiology
 Enterobacteriaceae
• Some Enterobacteriaceae are true pathogens
– Salmonella spp.
– Shigella spp.
– Yersinia spp.
– Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)
• Most members of the Enterobacteriaceae are
opportunistic or cause secondary infections of wounds,
the urinary and respiratory tracts, and the circulatory
system e.g. E. coli.
• Enterobacteriaceae divided into TWO main groups
according to action on LACTOSE
– Lactose Fermenters (LF)
• E. coli, Citrobacter, Klbesiella, Enterobacter
– Lactose Non-Fermenters (LNF)
Dr. Nabil El Aila
• Salmonella, Shigella, Proteus, Yersinia
Diagnostic Microbiology
 Identification of Enterobacteriaceae
• Gram stain
– All Enterobacteriaceae are Gram-negative rods
– Arranged in single

Dr. Nabil El Aila

Diagnostic Microbiology
 Identification of Enterobacteriaceae
Biochemical reactions
• Oxidase test

– All members of Enterobacteriaceae are oxidase negative

– Pseudomonas is oxidase positive

• O/F test

– All members of Enterobacteriaceae are O+/F+

– Pseudomonas is O+/F-

• Nitrate reductase

– All members of Enterobacteriaceae are nitrate reductase positive

– Pseudomonas is nitrate reductase negative

 Classification of Enterobacteriaceae

Enterobacteriaceae

Lactose fermenters
Non-lactose fermenter
E. coli, Citrobacter,
Salmonell, Shigella
Klebsiella, Enterobacter
Proteus, Yersinia

There are several selective and differential media used to

isolate distinguishes between LF & LNF

The most important media are:

MacConkey agar

Eosin Methylene Blue (EMB) agar

Salmonella Shigella (SS) agar

In addition to Triple Sugar Iron (TSI) agar
 Differentiation between LF & NLF by Growth on MacConkey agar

MacConkey agar is selective & differential medium for Enterobacteriaceae

MacConkey Agar
Contains

Bile salts Crystal violet Lactose Neutral red

pH indicator
Inhibit growth of G+ve bacteria Cause of differential Acidic: Pink

Cause of selectivity
Lactose feremnters Lactose non feremnters
Pink colonies colorless colonies
Classification of Enterobacteriaceae according to lactose
fermentation (growth on MacConkey Agar)
Enterobacteriaceae

Lactose Fermenters Lactose Non-Fermenters

No acid
Acid
Neutral red
Colorless colonies
Pink colonies

Escherichia coli
Salmonella spp
Klebsiella spp
Schigella spp
Enterobacter spp
Proteus spp
Citrobacter spp Dr. Nabil El Aila Yersinina spp
Diagnostic Microbiology
 Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on MacConkey agar
• Method:
– MacConkey agar is inoculated with tested organism using
streak plate technique
– Incubate the plate in incubator at 37 C/24 hrs
• Results:
– LF organism appears as pink colonies (e.g. E. coli)
– NLF organism appears as colorless colonies (e.g. Shigella)

Flame & Cool

1 2
3
4 Flame & Cool
5

Flame & Cool

 Growth of Enterobacteriaceae on
MacConkey agar

Colorless colonies Pink colonies

Uninoculated plate Lactose non feremters Lactose feremters

Salmonella, Shigella, E. coli, Citrobacter
Dr. Nabil El Aila
Proteus Klebsiella, Enterobacter
Diagnostic Microbiology
 Reaction on Salmonella Shigella (SS) agar
• SS agar is a selective & differential medium used for isolation of
Salmonella and Shigella
• The selective agents are bile salts, and brilliant green dye, which inhibit
gram-positive organisms
• The medium contains only lactose as a differential agent and thus
differentiates on the basis of lactose fermentation
• The formation of acid on fermentation of lactose causes the neutral red
indicator to make pink colonies
• Non lactose fermenting organisms are colorless on the medium
• SS agar contains sodium thiosulfate and ferric ammonium citrate
allows the differentiation of organisms that produce H2S
– Lactose fermenters, such as E. coli, have colonies which are pink
– Shigella appears transparent or amber
– Salmonella appears transparent or amber with black centers due to
H2S production

Lactose fermenter Neutral red

Lactose Acid Pink colonies
H2S + Ferric ammonium citrate Ferrous sulfide
Black precipitate
 Identification of Enterobacteriaceae
Differentiation between LF & NLF by Growth on SS agar

• Method:

– SS agar is inoculated with tested organism using

streak plate technique

– Incubate the plate in incubator at 37 C/24 hrs

Flame & Cool
1 2
3
4
Flame & Cool
5
Dr. Nabil El Aila
Flame & Cool
Diagnostic Microbiology
 Growth of Enterobacteriaceae on SS agar

A Klebsiella pneumoniae Both are lactose fermenters

B Escherichia coli
C Salmonella sp Both Salmonella sp. & Proteus product H2S
D Proteus mirabilis
E Ps. aeruginosa
Pseudomonas colonies are nearly colorless
 Reaction on Triple Sugar Iron (TSI) Agar
• TSI contains
– Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue,
and Yeast Enriched Peptone provide the nitrogen, carbon, and
vitamins required for organism growth.
– Three different types of sugars
• Glucose (1 part)
• Lactose (10 part)
• Sucrose (10 part)
– Phenol red (acidic: Yellow)
• TSI dispensed in tubes with equal butt & slant
 Reaction on Triple Sugar Iron (TSI) Agar

• Principle
– To determine the ability of an organism to attack a specific
carbohydrate incorporated into a basal growth medium, with
or without the production of gas, along with the determination
of possible hydrogen sulphide production.
• When the carbohydrates are fermented, acid production is detected
by the Phenol Red pH indicator.
• Sodium Thiosulfate is reduced to hydrogen sulfide, and hydrogen
sulfide reacts with an iron salt yielding the typical black iron sulfide.
• Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator.
Sodium Chloride maintains the osmotic balance of the medium.
Agar is the solidifying agent
 Reaction on TSI
• Method:
– Inoculate TSI medium with an organism by
inoculating needle by stabbing the butt and
streaking the slant
– Incubate at 37°C for 24 hours

Dr. Nabil El Aila

Diagnostic Microbiology
 Result
Reaction on TSI Example

Butt Slant Result

H2 S
color color
Non fermenter
Alk/Alk/-
Red Red Negative e.g.
(No action on sugars)
Pseudomonas
Negative A/Alk/- LNF
Yellow Red (Glucose fermented e.g. Shigella
without H2S)
Positive LNF
black in A/Alk/+ e.g. Salmonella &
Yellow Red butt (Glucose fermented Proteus
with H2S)

LF
A/A/-
Negative e.g. E. coli,
Yellow Yellow (three sugars are
Klebsiella,
fermented)
Enterobacter
 Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
Gram Oxidase Nitrate O/F MacCon SS EMB
stain reductase key
E. coli -ve rod -ve +ve O+/F+ LF LF Metallic
sheen
Citrobacter -ve rods -ve +ve O+/F+ LF LF Dark

Klebsiella -ve rods -ve +ve O+/F+ LF LF Dark

Enterobacter -ve rods -ve +ve O+/F+ LF LF Dark

Salmonella -ve rods -ve +ve O+/F+ NLF NLF/ Colorless

H2S
Shigella -ve rods -ve +ve O+/F+ NLF NLF Colorless

Proteus -ve rods -ve +ve O+/F+ NLF NLF/ Colorless

H2S
 Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
TSI Indole MR VP Citrate Urease Motility

E. coli A/A/- +ve +ve -ve -ve -ve Motile

Citrobacter A/A/- +ve +ve -ve +ve -ve Motile

freundii
Klebsiella A/A/- -ve -ve +ve +ve +ve Non
pneumoniae motile
Enterobacter A/A/- -ve -ve +ve +ve +ve Motile
cloacae
Salmonella A/Alk/+ -ve +ve -ve +ve -ve Motile
typhi
Shigella A/Alk/- -ve +ve -ve -ve -ve Non
boydii motile
Proteus A/Alk/+ -ve +ve -ve +ve +ve Motile
mirabilis Swarwing
 Lysine Iron Agar (LIA)
• Lysine iron agar (LIA) slants test organisms for the ability to
deaminate lysine or decarboxylate lysine. Lysine deamination is an
aerobic process which occurs on the slant of the media. Lysine
decarboxylation is an anaerobic process which occurs in the butt
of the media.
• LIA slants contain lysine, glucose, peptones, bromcresol purple (pH
indicator), sodium thiosulfate and ferric ammonium citrate. If the
organism has the ability to decarboxylate lysine, it produces an
amine end-product which reacts with the pH indicator to give a
purple color in the butt of the tube.
(Negative decarboxylation: yellow butt).
• If the organism has the ability to deaminate lysine, the ammonia
produced will react with the ferric ammonium citrate to produce a
dark red color on the slant of the tube. (Negative
deamination: purple slant). Organisms which produce hydrogen
sulfide gas will exhibit a black precipitate in the butt of the tube.
 Lysine Iron Agar (LIA)
• This agar is used as a diagnostic test for salmonellae.
• Salmonellae are the only group of Enterobacteriaceae that regularly
decarboxylate lysin (by lysine decarboxylase) and produce large
amounts of hydrogen sulphide.
• Bacteria that decarboxylate lysine cause an alkaline reaction (purple
colour) throughout the medium.
• Those that do not, produce an alkaline slant and an acid butt
(yellow) due to fermentation of glucose.
• Some bacteria like proteus species may deaminate the lysine and
produce a red slant and acid butt.
• Production of hydrogen sulphide causes a blackening in the
medium due to formation of ferrous sulphide. The indicator is
bromcresol purple.
 Tube 1: Positive decarboxylation (butt), negative deamination (slant)
Tube 2: Negative decarboxylation (butt), positive deamination (slant)

Dr. Nabil El Aila

Diagnostic Microbiology
 Lysine Iron Agar (LIA)

3. Alkaline/yellow/H2S:
Uninoculated medium Alkaline/alkaline/H2S:
Citrobacter.
Salmonella.
 COLLECTION, TRANSPORT, AND EXAMINATION
OF FAECES ( STOOL SPECIMENS )
Possible pathogens
Gram Positive Gram Negative
• Clostridium perfringens Shigella species
type A and C Salmonella species
• Clostridium difficile Campylobacter species
Bacillus cereus ( toxin ) Escherichia coli
• Staphylococcus aureus(toxin) ( ETEC,EIEC,EPEC)
Vibrio cholerae 01
Other Vibrio species
Yersinia enterocolitica

• Viruses : mainly rotaviruses, adenoviruses, coxsackieviruses, echoviruses, and

polioviruses.
• Fungi : Candida albicans .
Dr. Nabil El Aila
Diagnostic Microbiology
 Parasites :
Eggs : Amoebae :
Ascaris lumbricoides Entamoeba histolytica
Hookworm Flagellates:
Trichuris trichiura Giardia lamblia
Schistosoma species Trichomonas hominis
Hymenolepis species Ciliates :
Diphylobothrium latum Taenia species
Entrobius vermicularis Balantidium coli,
Larvae:
Strongyloides stercoralis
Cysts :
Entamoeba histolytica
Giardia lamblia
Balantidium coli
Isospora belli ( oocysts )
Commensals
The normal microbial flora of the gastrointestinal tract is greatly influenced by
diet. Microorganisms, which may form part of this normal flora, include:
Gram Positive Gram Negative
Enterococci Escherichia coli*
Anaerobic streptococci Proteus*
Lactobacilli Enterobacter*
Clostridia Hafnia *
Citrobacter*
Providencia*
Morganella*
Serratia*
Klebsiella *
Bacteroides species
Pseudomonas aeruginosa
• These genera belong to the family Enterobacteriaceae. Enterobacteria are often
described as coliforms.
• Fungi: Candida species and yeasts.
• Other: Mycoplasma and a variety of protozoa.
 Collection of stool sample
• Stool specimens are usually collected in a clean, dry disinfectant free
bedpan or suitable wide necked container.
• The container need not be sterile, ask the patient to avoid
contaminating the stool with urine.
• Transfer a portion of the specimen especially which contains mucus,
pus or blood into a clean dry leak-proof container.
• A diarrheal stool usually gives good results.
• Label the specimen, on the container not lid, and send it with a request
form to reach the laboratory within 1 hour.
• Stool passed into the toilet bowel must not be used for culture.
• No toilet paper should be placed in the bed pan or specimen container,
which may contain bismuth that interferes with the laboratory tests.
 Collection of stool sample
• If it is not possible to obtain stool specimen, a rectal swab may be used
to obtain the sample by inserting a cotton swab into the anus beyond
the anal sphincter for about 10 seconds, carefully rotate the swab and
withdraw.
• If delay over 18-24 hours is suspected, the specimen should be mixed
with an equal volume of buffered glycerol saline. Also you can insert
the swab in a container of sterile cary - Blair transport medium.
• Salmonella, Shigella , Vibrio and Yersenia species survive well in cary-
Blair medium for up to 48 hours but Campylobacter for up to 6 hours.
• If cholera is suspected transfer about 1ml of specimen into 10ml of
sterile alkaline peptone water , label and send to the microbiology
department within 8 hours of collection.
Dr. Nabil El Aila
Diagnostic Microbiology
 Suspected organisms
1- E-coli (infant &NLF)
2- salmonella & shigella
3- campylobactor
jejuni most common human pathogen
indol&citrate (+)
urease (- )
oxidase (+)
nalidixic acid (S)
cephalo thin (R)
Dr. Nabil El Aila
Diagnostic Microbiology
 Suspected organisms
• 4- V.cholerae
serogroup -01
catalse (+)
oxidase (+)
motile (+)
lactose (NLF)
sensitive to (dry – sunlight – acid pH)
TCBS (thiosulfate citrate bile sucrose
Agar – yallow colonies)
 Stool or Rectal swab culture
*MacConky plate (NLF)

*XLD agar
*HE
*selenite F broth/ GN broth

*TCBS (y.colores)
*alkaline peptone water (APW)

*Selective media for campylobactor

(skirrow &blaser with suplements)
campylobactor microaerophilic
(42C°-O2 5%-Co210%-N2 85%-48hr)
 Detection of
Salmonella & Shigella

Stool/Rectal swab

Direct Culture SS agar Incubate Overnight 37C° GN/Selenite broth

Check for suspected colonies

Subculture Hecktoen

Incubate Overnight 37C°

Check for suspected colonies

Confirmation using Biochemical and serological reaction

 Salmonella on SS-agar: Salmonella on Hektoen agar:

Salmonella on XLD Gram stain of Salmonella

 Bichemical reactions of
Salmonella on Triple Sugar Iron

Bichemical reactions of
Salmonella on API20E
Dr. Nabil El Aila
Diagnostic Microbiology
 Salmonella on SS-agar: Salmonella on Hektoen agar:

Salmonella on XLD Gram stain of Salmonella

 Bichemical reactions of
Salmonella on Triple Sugar Iron

Dr. Nabil El Aila

Diagnostic Microbiology
 Shigella on Hektoen agar:
Shigella on SS-agar:

Gram stain of Shigella

Shigella on XLD
Dr. Nabil El Aila
Diagnostic Microbiology
 API20E

Dr. Nabil El Aila

Diagnostic Microbiology
 Detection of Vibrio
Cholera

Stool/Rectal swab

Direct Blood agar Direct TCBS Alkaline peptone water

Incubate Overnight 37C°

Check for suspected colonies Subculture TCBS

Incubate Overnight 37C°

Check for suspected colonies

Confirmation using Biochemical and serological reaction

Dr. Nabil El Aila
Diagnostic Microbiology
 Dr. Nabil El Aila
Vibrio cholerae TCBS
Diagnostic Microbiology
 Enterobacteriaceae
Biochemical reactions
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University

Dr. Nabil El Aila

Diagnostic Microbiology
 Lactose Fermentation
• MacConkey Agar contains bile salts and crystal violet, both
inhibitory to Gram-positive bacteria and selects Gram-negative
bacteria, such as E. Coli.
• It also differentiates lactose-fermenting bacteria, such as E.
Coli from non-lactose fermenting bacteria.
• Media and Reagent: MacConkey Agar and neutral red dye
• Method: Streak MAC plate and incubate at 37°C for 2 days.
• Expected results:
– Positive test: Lactose fermentation = Growth and color
change to pink
– Negative test: No lactose fermentation = May or may not
grow and no color change
Dr. Nabil El Aila
Diagnostic Microbiology
 Results of Lactose Fermentation

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC Test
• Indole, Methyl Red, Voges-Prosakaur, Citrate
(IMViC) Tests:
– The following four tests comprise a series of
important determinations that are collectively
called the IMViC series of reactions
– The IMViC series of reactions allows for the
differentiation of the various members of
Enterobacteriaceae.

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: Indole test

Principle

Certain microorganisms can metabolize
tryptophan by tryptophanase

The enzymatic degradation leads to the
formation of pyruvic acid, indole and ammonia

The presence of indole is detected by addition
of Kovac's reagent.

Tryptophanase
Tryptophane Indole + Pyurvic acid + NH3
amino acids Kovac’s Reagent

Dr. Nabil El Aila Red color in upper organic layer`

Diagnostic Microbiology
 IMViC: Indole test
 Method:
 Inoculate the test organism into tryptophane
broth
 Incubate at 37°C for 24 hours
 After incubation interval, add 1 ml Kovacs
reagent which contain 4 (p) – dimethylamino
benzaldehyde, shake the tube gently and read
immediately

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: Indole test
Negative test Positive test
e.g. Klebsiella e.g. E. coli
 Result:
 A bright pink color in the top
layer indicates the presence of
indole
 The absence of color means that
indole was not produced i.e.
indole is negative

 Significance:
 Used in the differentiation of
genera and species. e.g. E. coli (+)
from Klebsiella (-).

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC test
Methyl Red-Voges Proskauer (MR-VP) Tests
• Different bacteria convert dextrose and glucose to pyruvate
using different metabolic pathways.
• Some of these pathways produce unstable acidic products
which quickly convert to neutral compounds.
• Some organisms use the butylene glycol pathway, which
produces neutral end products, including acetoin and 2,3-
butanediol.
• Other organisms use the mixed acid pathway, which produces
acidic end products such as lactic, acetic, and formic acid. These
acidic end products are stable and will remain acidic.
 IMViC test
Methyl Red-Voges Proskauer (MR-VP) Tests
Principle
Glucose

Acidic pathway
Or Neutral pathway

Acety methyl carbinol

Mixed acids (ACETOIN)
 pH less than 4.4
Barrit’s A (α-naphthol)
Methyl Red Barrit;s B (40% KOH)
indicator

MR positive VP positive
Pink color
Red color E. coli Klebsiella
 IMViC: Methyl red
Principle:
• Methyl red test is used to identify enteric bacteria based on
their pattern of glucose metabolism.
• If they use mixed acid pathway and produce acidic products,
then they are called methyl-red-positive.
• If they use butylene glycol pathway and produce neutral end
products, then they are called methyl-red-negative

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: Methyl red
• Method:
• Inoculate 10ml portion of the MR-VP medium and incubate at
37°C for 2-5 days.
• After incubation, transfer 2.5 ml of inoculate to another tube
and add five drops of methyl red.
• Roll between the palms of hands to disperse methyl red.

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: Methyl red
• Results:
– Positive test: acids + methyl red = red solution
– Negative test: neutral end products + methyl red =
yellow color
• Significance:
This test is used to differentiate
Enterobacteriacaceae species
espcially E. coli and E. aerogens

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: VOGES-PROSKAUER TEST

Principle:
• It is used to identify enteric bacteria based on their pattern of
glucose metabolism.
• The enterics that produce neutral end-products, such as acetoin
are detected by VP test.
• Its presence is used as indicator of 2,3 butylene glycol
Fermentation
• The detection of acetoin in alkaline pH is accomplished by
alpha-Naphthol reagent.

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: VOGES-PROSKAUER TEST

• Method:
• Inoculate medium and incubate at 37°C for 48 hours.
• After incubation, transfer 2.5 ml of inoculate to another tube
and add six drops of Barritt’s Reagent A (contains alpha-
naphthol) and two drops of Barritt’s Reagent B(contains
KOH).
• Gently mix and let it sit for 10-15 minutes to allow time for
color development.

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: VOGES-PROSKAUER TEST

• Results:
– Positive test: acetoin + alpha-naphthol + KOH = red color
– Negative test: alpha-naphthol +KOH = copper color
• Significance:
This test is used to differentiate
Enterobacteriacaceae species
espcially E. coli and E. aerogens

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: CITRATE TEST
Principle:
Citrate Pyruvate CO2 + Na + H2O Na2CO3

Alkaline,↑pH
Simmone’s Citrate media
Contains Citrate as a sole of C source

Bromothymol blue

Positive test

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: CITRATE TEST
• Principle:
• Citrate is an organic molecule that can be utilized by bacteria
that produce the enzyme citrase.
• Citrase is produced by some bacteria such as E. aerogenes but
not by others like E. Coli
• Method:
• Inoculate the test organism onto a slant containing Simmon
Citrate agar.
• Simmon’s Citrate Agar contains sodium citrate (carbon
source), & pH indicator—bromthymol blue.
• Incubate at 37°C for 24 hours.

Dr. Nabil El Aila

Diagnostic Microbiology
 IMViC: CITRATE TEST
• Results:
– Positive test: Growth and color changes to blue
– Negative test: No growth and color remains green
• Significance:
• This test is used to help differentiate
species of the family Enterobacteriaceae.
• It is selective for bacteria that has
the ability to consume citrate as
its sole source of carbon

Dr. Nabil El Aila Positive Negative

Diagnostic Microbiology
Principle
Urease Test
• Urea agar contains urea and phenol red
• Urease is an enzyme that catalyzes the conversion of urea
to CO2 and NH3
• Ammonia combines with water to produce ammonium
hydroxide, a strong base which ↑ pH of the medium.
• ↑ in the pH causes phenol red r to turn a deep pink. This is
indicative of a positive reaction for urease
Urease H2O
Urea CO2 + NH3 NH4 OH ↑ in pH

Phenol Red

Method Pink
Positive test
 Streak a urea agar tube with the organism
 incubate at 37°C for 24 h
 Urease Test
• Result
• Positive test: production of alkaline
end products = pinkish red color
• Negative test: No color change
• Significance:
• Differentiate salmonella and shigella
which are urease negative from
urease positive Non pathogens.
• Proteus, klebsiella and some
citrobacter species are urease
positive
Negative test Positive test
• Helicobater pylori is also
Urease positive
Dr. Nabil El Aila
Diagnostic Microbiology
 Motility Test
• Principle:
• Motility Test Media is a semi-solid agar designed to
demonstrate motility by diffusion.
• This is not a biochemical test, but it can distinguish bacteria. It
determines presence of flagella.
• Method:
• Inoculate a semi-solid nutrient medium by stabbing 2 cm
into the center of the medium
• Inoculate at 37C° for 24-48 hours.

Dr. Nabil El Aila

Diagnostic Microbiology
 Motility Test
• Expected results:
– Positive test: Growth spread away from the line of
inoculation = motile
– Negative test: Growth only occurred at the line of
inoculation = Non-motile
• Significance:
• This test is used for the differentiation
of microorganisms on the basis of
motility.

Dr. Nabil El Aila

Diagnostic Microbiology
 H2S Production
• Principle:
• Bacteria use enzyme cysteine desulfurase to hydrolyze the amino
acid cysteine, forming hydrogen sulfide as end-product.
• To test for hydrogen sulfide production, a medium with a sulfur-
containing compound and iron salts is inoculated and incubated. If
the sulfur is reduced and hydrogen sulfide is produced, it will
combine with the iron salt to form a visible black ferric sulfide (FeS)
in the tube
• Media and Reagent: SIM tube (sulphide, Indole and Motility) with
cysteine and ferrous sulfate (detects H2S)
• Method: Inoculate the media and incubate at 37°C for 24-48 hours.

Dr. Nabil El Aila

Diagnostic Microbiology
 H2S Production
• Expected Results:
– Positive Test: H2S production = Black
– Negative Test: No H2S production = No blackening of medium
• Significance:
• This test is used to determine the ability
to reduce sulfur into H2S.
• Differentiate species of the family
Enterobacteriaceae.
• Identifying unknown organisms such as
certain Proteus and salomenella

Dr. Nabil El Aila Negative Positive

Diagnostic Microbiology
 Description:
 Aspergillosis is a spectrum of diseases of humans
and animals caused by members of the genus
Aspergillus.
 These include :
 (1) mycotoxicosis
 (2) allergy
 (3) colonisation without extension .
 (4) invasive, inflammatory, granulomatous,
narcotising disease of lungs, and other organs.
 (5) systemic and fatal disseminated disease.
 Clinical manifestations:

 1. Pulmonary Aspergillosis: including allergic,

aspergilloma and invasive aspergillosis
 2. Disseminated Aspergillosis
 3. Aspergillosis of the paranasal sinuses:
 4. Cutaneous Aspergillosis:
 Clinical material: Sputum, bronchial washings
and tracheal aspirates from patients with
pulmonary disease and tissue biopsies from
patients with disseminated disease.
1. Direct Microscopy: (a) Sputum, washings and
aspirates make wet mounts in either 10% KOH &
Parker ink or Calcofluor and/or Gram stained smears;
2. (b) Tissue sections should be stained with H&E, GMS
and PAS digest.
• Note Aspergillus hyphae may be missed in H&E
stained sections.
• Examine specimens for dichotomously branched,
septate hyphae.
 The presence of hyaline, branching septate hyphae,
consistent with Aspergillus in any specimen, from a
patient with supporting clinical symptoms should be
considered significant. Biopsy and evidence of tissue
invasion is of particular importance.
 * Remember direct microscopy or histopathology does
not offer a specific identification of the causative agent.
 Clinical specimens should be inoculated onto
primary isolation media, like Sabouraud's
dextrose agar. Colonies are fast growing and
may be white, yellow, yellow-brown, brown to
black or green in colour
 Interpretation: Aspergillus species are well
recognised as common environmental airborne
contaminants, therefore a positive culture from
a non-sterile specimen, such as sputum, is not
proof of infection.
 Immunodiffusion tests for the detection of
antibodies to Aspergillus species have
proven to be of value in the diagnosis of
allergic, aspergilloma, and invasive
aspergillosis. However, they should never
be used alone, and must be correlated with
other clinical and diagnostic data.
 Aspergillus flavus,

 Aspergillus fumigatus

 Aspergillus nidulans

 Aspergillus niger

 Aspergillus terreus
 Clinical significance: Aspergillus flavus has a world-
wide distribution and normally occurs as a saprophyte
in soil and on many kinds of decaying organic matter.
A. flavus is the second most common species (next to A.
fumigatus) to be isolated from human infections, and it
is often associated with invasive aspergillosis seen in
immunosuppressed patients and in paranasal sinus
infections.
 On Czapek dox agar, colonies are granular, flat, often
with radial grooves, yellow at first but quickly
becoming bright to dark yellow-green with age.
 Conidial heads are typically radiate, mostly 300-400
um in diameter, later splitting to form loose columns,
biseriate but having some heads with phialides borne
directly on the vesicle.
 Conidiophores are hyaline and coarsely roughened,
the roughness often being more noticeable near the
vesicle.
 Conidia are globose to subglobose (3-6 um in
diameter), pale green . Some strains produce brownish
sclerotia
 Aspergillus fumigatus is truly a global mould and
has been found almost everywhere on every
conceivable type of substrate, especially soil and
decaying organic debris. A. fumigatus is an
important human pathogen and it is the most
common cause of all forms of invasive and non-
invasive aspergillosis.
 On Czapek dox agar, colonies show typical blue-green
surface pigmentation .
 Conidial heads are typically columnar (up to 400 x 50
um but often much shorter and smaller) and
uniseriate.
 Conidiophores are short, smooth-walled and have
conical-shaped terminal vesicles which support a
single row of phialides on the upper two thirds of the
vesicle.
 Conidia are produced in long chains and are globose
to subglobose (2.5-3.0 um in diameter), green and
rough-walled.
 Clinical significance:

 Aspergillus nidulans is a typical soil fungus with

a world-wide distribution. It has also been
reported as a causative agent of aspergillosis in
humans and animals.
 On Czapek dox agar, colonies are typically plain green in color .

 Reverse may be olive to drab-gray or purple-brown.

 Conidial heads are short, columnar (up to 70 x 30 um in

diameter) and biseriate.

 Conidiophores are usually short, brownish and smooth-walled.

 Conidia are globose (3.0-3.5 um in diameter) and rough-walled

 Clinical significance:

 This is the third most common species

associated with invasive pulmonary
aspergillosis. It is also often a causative agent
of aspergilloma and is the most frequently
encountered agent of otomycosis. A. niger may
also be a common laboratory contaminant.
 On Czapek dox agar, colonies consist of a compact white or yellow basal felt
covered by a dense layer of dark-brown to black conidial heads.

 Conidial heads are large (up to 3 mm x 15-20 um in diameter), globose, dark

brown,and becoming radiate .

 Conidiophores are smooth-walled, hyaline or turning dark towards the vesicle.

 Conidial heads are biseriate with the phialides borne on brown, often septate
metulae.

 Conidia are globose to subglobose (3.5-5.0 um in diameter), dark brown to black

and rough-walled.
 Clinical significance:

 Aspergillus terreus occurs commonly in soil and

is occasionally reported as a pathogen of
humans and animals.
 On Czapek dox agar, colonies are typically cinnamon-
buff to sand brown in color with a yellow to deep dirty
brown reverse.
 Conidial heads are compact, columnar (up to 500 x 30-
50 um in diameter) and biseriate.
 Conidiophores are hyaline and smooth-walled.
 Conidia are globose to ellipsoidal (1.5-2.5 um in
diameter), hyaline to slightly yellow and smooth-
walled