Biochemical

tests
 I-SIM medium
used to determine :
1- sulfur reduction
2- indole production from tryptophan
3- motililty
SULFUR
production
By cysteine
desulfurase enz.
which Thiosulfate reductase
catalyzeputrefaction enz., that reduce sulfur
of cysteine to (in thiosulfate form)
pyruvate Sodium thiosulfate H2S
Cysteine +H2O+H pyruvate

Both produce H2S which combine with iron found in media FeS (black)
 INDOLE production

Tryptophane in media hydrolyzed by tryptophanase enz. To pyruvate where

ammonia and indole produced that indicated by Kovac’s reagent (this regent
contain dimethylamino benzaldhyde (DMABA) react with indole and give red
color (+ve results).
Motility

Tested organism inoculated with single stab needle, motile organisms move
through semisolid media that detected by radiating growth in all directions
and give fuzzy appearance.
Sulfur reducing bacteria are: Salmonella and Proteus
Non-sulfur reducing : Morgonella
All Enterobacteriaceae are motile
2- OF (Oxidation-Fermentation) Test
Carbohydrates are organic molecules that contain carbon, hydrogen and oxygen in the ratio
(CH2O)n. Organisms use carbohydrate differently depending upon their enzyme complement.
Objective
To detect the oxidation or fermentation of carbohydrates by bacteria.
Principle
Whether an organism is oxidative or fermentative can be determined by using Hugh and
Leifson’s medium, which contain tryptone and bromothymol blue (an indicator). One of the
sugars, such as glucose, xylose, mannitol, lactose, sucrose, and maltose is added to the medium
which serves as the fermentable carbohydrate.
An organism is inoculated to two tubes of each OF Medium. Once inoculated, one tube is
overlaid with mineral oil or melted paraffin producing an anaerobic environment. The other
tube is left open to the air. Growth of microorganisms in this medium is either by utilizing the
tryptone which results in an alkaline reaction (dark blue color) or by utilizing glucose, which
results in the production of acid (turning bromothymol blue to yellow).
Oxidative utilization of the carbohydrate will result in acid production (yellow) in the open tube
only. Fermentative utilization of the carbohydrate will result in acid production (yellow) in both
the open and closed tubes. Acidic changes in the overlaid tubes are considered to be a result of
true fermentation, while acidic development in the open tubes are due to oxidative utilization
of the carbohydrate present. Asaccharolytic organisms will not produce acid in either tube.
Media:
Hugh and Leifson’s medium: Peptone 2.0gm/L, Sodium chloride
5.0gm/L, Dipotassium phosphate 0.30gm/L, Glucose (Dextrose)
10.0gm/L, Bromothymol blue 0.030gm/L, Agar 3.0gm/L, Final pH ( at 25°C)
7.1±0.2.
Method:
Obtain pure, isolated colonies from an 18-24 hour culture.
For each test organism, inoculate tubes in duplicate. Inoculate by
stabbing the agar to approximately 1/4 inch from the bottom.
Apply sterile mineral oil, sterile melted paraffin, or sterile melted
petrolatum to one of each duplicate tubes. Tighten the cap of the
overlaid tube, and loosen the cap of the non-overlaid tube.
Incubate both tubes aerobically at 35ºC. for up to 14 days.
Examine tubes daily for color change.
Expected Results
Positive: A positive carbohydrate utilization test is indicated by the development
of a yellow color in the medium.
Oxidative :Development of a yellow coloration in the open tube only.
Fermentative: Development of a yellow coloration in both open and closed
tubes.
Negative: A negative carbohydrate utilization test is indicated by the absence of a
yellow color (media remains green or turns blue).
Non-oxidizer/Non-fermenter
3. Voges-Proskauer ( VP) test
Principle of Voges–Proskauer (VP) Test
The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethyl
carbinol from glucose fermentation. If present, acetylmethyl carbinol is converted to diacetyl in
the presence of ∝- naphthol, strong alkali (40% KOH), and atmospheric oxygen. The ∝-
naphthol was not part of the original procedure but was found to act as a color intensifier
by Barritt and must be added first. The diacetyl and quanidine-containing compounds found in
the peptones of the broth then condense to form a pinkish red polymer.
2 pyruvate = acetoin + 2CO2
acetoin + NADH + H+ = 2,3-butanediol + NAD+
Media and Reagents used in Voges–Proskauer (VP) Test
Peptone 7g Voges-Proskauer Reagent A: Barritt’s reagent A
Glucose 5g Alpha-Naphthol, 5% 50 gm
Dipotassium phosphate 5g
Absolute Ethanol 1000 ml

Voges-Proskauer Reagent B: Barritt’s reagent B

Potassium Hydroxide 400 gm
Deionized Water 1000 ml
Method of Voges–Proskauer (VP) Test
Prior to inoculation, allow medium to equilibrate to room temperature.
Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium.
Incubate aerobically at 37 degrees C. for 24 hours.
Following 24 hours of incubation, aliquot 2 ml of the broth to a clean test tube.
Re-incubate the remaining broth for an additional 24 hours.
Add 6 drops of 5% alpha-naphthol, and mix well to aerate.
Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
Observe for a pink-red color at the surface within 30 min. Shake the tube vigorously during the 30-min
period.
Positive Reaction: A pink-red color at the
surface
Examples: Viridans group streptococci
(except Streptococcus vestibularis), Listeria,
Enterobacter, Klebsiella, Serratia
marcescens, Hafnia alvei, Vibrio
eltor, Vibrio alginolyticus, etc.
Negative Reaction: A lack of a pink-red
color
Examples: Streptococcus mitis, Citrobacter
sp., Shigella, Yersinia, Edwardsiella,
Salmonella, Vibrio furnissii, Vibrio fluvialis,
Vibrio vulnificus, and Vibrio
parahaemolyticus etc.
4. Nitrate reduction test
Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen (O2). Many gram-
negative bacteria use nitrate (NO3) as the final electron acceptor.
Nitrate reduction test is a test that determines the production of an enzyme called nitrate reductase, which
results in the reduction of nitrate (NO3).
Bacterial species may be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous
gases.
Principle:
A heavy inoculum of test organism is incubated in a broth containing nitrate (NO3). The organisms capable of
producing the nitrate reductase enzyme reduce the nitrate, present in the broth, to nitrite (NO2) which may
then be further reduced to nitric oxide (NO), nitrous oxide (N2O), or nitrogen.
The nitrate reduction test is based on the detection of nitrite (NO2) and its ability to form a red compound
when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid) which then reacts with a α-
naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye.

However, only when nitrate is present in the medium, red color will be produced. If there’s no red color in
the medium after you’ve added sulfanilic acid and α-naphthylamine means only that nitrite is not present in
the medium.
There is two explanations for this observation.
1. The nitrate may not have been reduced; the strain is nitrate-negative.
2.The nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide,
nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite; the strain is nitrate-
positive.
Media:
Nitrate Broth
Peptone 5 g/l, Meat extract 3 g/l, Potassium nitrate 1 g/l.
Final pH 7.0 ± 0.2 at 25°C
Method
Determination of nitrate reduction to nitrite is a two step process. First, the reduction of nitrate to nitrite is
determined by the addition of Nitrate Reagents A and B, then if necessary, the reduction of nitrate beyond
nitrite is determined by the addition of Nitrate Reagent C (zinc dust).
Inoculate the nitrate broths with bacterial suspension.
Incubate the tubes at the optimal temperature 30°C or 37°C for 24 hours.
After incubation look for N2 gas first before adding reagents.
Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B.
Observe for the reaction (color development) within a minute or less.
If no color develops add zinc powder.
Observe for at least 3 minutes for a red color to develop after addition of zinc.
Expected Results
Positive Test:
– Development of a cherry red coloration on addition of reagent A and B
– Absence of a red color development on adding Zn powder
Negative Test:
– A development of red color on addition of Zn powder
5. Coagulase test
Staphylococcus aureus act as opportunistic pathogen highly resist
immunosystem and antimicrobial agents due to coagulase enz. which
conjugate with plasma components and form fibrin barriers around bacterial
cells protect from phagocytosis or any attack.
There are two types of coagulase:
A-free coagulase
Extracellular enz. That released from cell and react with plasma component called
coagulase reacting factor (CRF) an form coagulase-CRF complex that convert
fibrinogen to fibrin clot.
B-Bound coagulase
(clumping factor) that attached to bacterial cell wall and react directly with fibrinogen
in plasma, so, making fibrinogen ppts and cells clump together.
Tests for coagulase determination:
1- Test tube
Used to detect both free and bound coagulase, by adding tested organism in test tube
with rabbit plasma if coagulation occurred within 24 hour without shaking so, +ve
results.
2- Slide test
Detect only bound coagulase, bacteria added to slide containing small amount of
plasma and agglutination occur within 1-2 minutes ( so bounded coagulase).
2- Slide test
Used to detect bounded coagulase only, bacterial cells added to
slide containing small amount of rabbit’s plasma, if agglutination
occur within 1-2 minutes so, bounded coagulase present.
6. Triple Suger Iron (TSIA) /Klingler Iron Agar (without sucrose)
Both used to differentiate members of Enterobacteriaceae and to
distinguish them from other Gram-ve bacteria
TSIA is a rich medium used to differentiate bacteria on basis of :
Glucose fermentation 10g
Sucrose fermentation 10 g
Lactose fermentation 10 g
Sulfur reduction
Media contain three types of (sugars, animal proteins as nitrogen source,
ferrious sulphate and sodium thiosulphate as source of oxidized
sulphur, phenol red as pH indicator).
1- when media inoculated with glucose only fermenter the acids products
will lower pH of media and phenol red convert to yellow color (bottom
only) in few hours.
As glucose present in low amount so it will exhausted in 12 hours,
organism in slope part will utilize aminoacids in above part and give
ammonia that increase pH in upper part and give red color at slope.
 Red slant/ yellow bottom (K/A) Glucose fermented only and proteins utilized aerobically and alkalin
produced in slant
2- when organism ferment three sugars whole media (slope and bottom convert yellow) due
to a lot of acids produced.

Yellow slant/yellow bottom (A/A)

Triple sugars fermented
Yellow slope/yellow bottom (KIA) glucose and lactose fermented with acid
accumulation (no sucrose)
Yellow slope/yellow bottom (TSIA) glucose and lactose and / or sucrose fermented with
acid accumulation
3- Orgaism that reduce sulfur and produce H2S
For thiosulphate reduction it occurred in acidic condition to be give black color with iron,
so black color in bottom indicate that glucose utilized and fermented.
Yellow slope and black bottom, H2S produced, glucose and lactose and /or sucrose
fermented
Red slope and black bottom, H2S produced and glucose only fermented
4- Detachment (cracks) of agar indicate carbohydrate fermentation and gas
production.
Red slope/red bottom (K/K ) no fermentation, protein utilized aerobic and anaerobic (not
from Enterobacteriaceae.

Red slope/no change bottom (K/NO) no fermentation, protein utilized aerobic only (not from
Enterobacteriaceae.
No change slope/no change bottom (NO/NO) no growth , no protein utilization (not from
Enterobacteriaceae.
4- Antimicrobial susceptibility test
Are substances control bacterial growth and this test used to identify antibiotic needed for
treatment of bacteria as some bacteria have responses toword some agents.
Disc diffusion method used for this puropse:
Then determine MIC for each type,
 7- Bacitracin , Novobiocin and Optochin and susceptibility
1- Bacitracin:
produced by Bacillus licheniformis , act as powerful peptide antibiotic
that inhibit bacterial cell wall synthesis.
Effective only for bacteria that have cell wall in growing process.
10 mm clear zone or greater said to be bactitracin inhibited.
2-Novobiocin:
Produced by Streptomyces niveus, interfere with ATPase activity.
16 mm clear zone or greater said to be novobiocin inhibited and used to
differentiate Staphylococcus saprophyticus (resistant) and
Staphylococcus epidermidis (susceptible)
3- Optochin
Interferes with ATPase activity.
19 mm clear zone or greater said to be optochin inhibited.
Used to differentiate Streptococcus pneumonia and Streptococcus
alpha-hemolytic)
8- Decarboxylation and deamination
decarboxylation: remove carboxyl group from amino
acids {COOH}
deamination: remove amino group from amino acids {-NH2}
A. Decaroxylation test
Mollers’s Decarboxylase Base Medium with peptone, glucose
and Bromocresol purple (purple 6.8 and above, yellow 5.2 or
below) and pyridoxyl phosphatase coenzyme.
After inoculation, oil added to prevent atmospheric oxygenand
fermentation occur.
Glucose fermentation Yellow color

After utilization of glucose, utilization of amino acids lead to

alkaline end product accumulation (NH3) Purple color
9. Deamination test
Microorganisms that produce phenylalanine deaminase enzyme used for
identify ability of organism to remove NH2group from phenylalanine.
O2
phenylalanine NH3+ pyruvic acid
Phenylalanine
deaminase FeCL3

Dark green
Chang color to deep or dark green indicate +ve results
10- PYR test
Strep0tococci (S.pyogenes) and Enterococci produce L-Pyrrolidonyl arylamidase that
hydrolyze the amide group of pyroglutamyl-B-naphthylamide to produce L-
pyrrolidone and B-naphthylamide which are colorless.

• After rapid incubation (18 hour) ,0.01% p-dimethylamino-cinnamaldhyde

(reagent) solution is added , deep red color appeared within few
minutes.
• Yellow or orange color indicate –ve results.

Heavy inoculum
11- Bile Esculin Agar

Act as selective and differential medium which test ability of microorganisms to hydrolyze Esculin in
presence of high bile content is usually identify Enterococcus faecalis

Differential by presence of
Esculin which is glycosidic
Selective by presence of bile
salts that inhibit growth of all G+ve
coumarin if organism can
hydrolyze esculin in presence of
bile salt esculetin formed
.
Where escletin formed react with ferric citrate present in medium form phenolic iron complex
turn to dark black color
Principle: G+VE and some streptococci are inhibited by high bile salt content present in media
Media
Bile esculin agar is a selective and differential medium which is used to
presumptively identify enterococci and group D streptococci based on the ability
of an organism to hydrolyze esculin.
Beef extract (11 g), enzymatic digest of gelatin (34.5 g), esculin (1 g), ox bile (2 g),
ferric ammonium citrate (0.5 g), agar (15 g), per 1000 mL, pH 6.6.
Method
• Inoculate one to two colonies from an 18- to 24-hour culture onto the surface of
the slant.
• Incubate at 35°-37°C in ambient air for 48 hours.
• Observe for growth and blackening of the medium
Expected results
Postive results dark black color
Negative light brown
12. DNA HYDROLYSIS TEST
DNA is large polymer of nucleotides act to be large to enter cell,and to utilize
DNA externally bacteria secret (DNAse) outside cells to hydrolyze this external
DNA to nucleotides and phosphate
These nucleotides move to cell membrane via protein transportation to utilized
and use these nucleotides as P,C,N sources
Principle:
Using DNAse agar that containing nutrients, DNA and methyl green as indicator
which bind with –ve charge DNA of phosphate used to determine ability of
organism to hydrolyze DNA or not.

1. Without indictor: In DNase agar without indicator, the hydrolysis of DNA is observed by a
clearing of the agar after addition of HCL (oligonucleotides dissolves in acid but DNA salts
are insoluble). The acid precipitates unhydrolyzed DNA making the medium opaque.
Therefore, DNase producing colonies hydrolyze DNA and produce a clear zone around the
growth.
2. With indicator: using methyl green color appeared when DNA molecule combine with methyl
green
Media:
DNase Medium
Pancreatic digest of casein (10 g), yeast extract (10 g), deoxyribonucleic
acid (2 g), NaCl (5 g), agar (15 g), methyl green (0.5 g), pH 7.5.
Method
Using a sterile loop, inoculate the DNase agar with the organism to be
tested on the test area.
Incubate the plate at 35-37°C for 24 hours.
After incubation observe the color change in DNase with methyl green.
Expected Results
Positive: Medium is colorless around the test organism.
Negative: If no degradation of DNA occurs, the medium remains green