BT233 – General Microbiology

Laboratory

Biochemical Tests

Next Week Lab(The last one) : will be online: Biochemical II

Identification Approach:
Genotypic, chemotaxonomic, and phenotypic methods of
characterizing the bacteria in order to find its taxonomic
position..
Colonial, cellular, and biochemical characterization is needed.
 Biochemical tests:
The tests that are performed on bacteria for their identification on
the basis of their biochemical activities towards different
biochemical compounds.

Each bacteria has which is called biochemical fingerprints which is a properties controlled by
the cells' enzymatic system, these includes bioenergetics, biosynthesis, and
biodegradation.
Enzymes aredivided according to where it works into (Intracellular and Extracellular).

Intracellular
(Work inside the bacteria to break down the nutrients to yield energy). Ex. All enzymes
involved in fermentation, glycolysis, and Krebs cycle.

Extracellular
(Synthesized in bacteria and secreted outside the cell to convert the high molecular compounds
to low one) Ex. Amylase and Maltase
Intracellular Enzymes : Carbohydrate Fermentation

The differences between respiration and fermentation?

Respiration (either Aerobic or Anaerobic):in aerobic respiration use 02as a final electron
acceptor. In Anaerobic respiration use inorganic ions asa final electrón Aceptore.g.NO-3or SO-4

Fermentation:inbiooxidationsuse organic substances as a final electron Acceptor and need no

oxygen.
Test # 1: Triple Suger Iron Agar ( TSI )
Principle:To determine the ability of bacteria to ferment a specific carbohydrate incorporated into a growth medium, with or
without the production of gas, along with the determination of possible hydrogen sulphide production.

This media contains:

• Three different types of sugars : lactose and sucrose1.0% , and glucose 0.1%

• pH indicator: Phenol red Neutral : Red

• Sulfur source: Sodium thiosulate or cysteine

• Sulfur indicator: Ferrous sulfate ( FeS04 )
• Carbon 'source : Peptone
TSI Slant Agar Inoculation (Only Gram-Negative Bacteria should be used)

TSI slant agar

By sterilely cooled needle inoculate the butt of the TSI at “ 1 " from your pure culture. Then from the same
culture inoculate the surface by sterile cooled loop through streaking. Incubate at 37°C for not more than 18
hours.
Bacteria if have enzymes to degrade both glucose, sucrose, lactose and peptone they start with the simplest then more
and more complex as in the following curve:

Slant (Aerobic Reaction)

Butt (Anaerobic Reaction)

If the reaction under anaerobic at X3 the reaction under aerobic must be at X4, If the
reaction under anaerobic at X4 the reaction under aerobic must be already completed.
Results :
1-If there is no fermentation occurred = Red color
2-If bacteria ferment lactose and sucrose = Yellow
3-If bacteria ferment glucose = Yellow
4-If bacteria catabolize pepton = Pink
5-If bacteria produce gas from fermentation = cracks in the butt.
6- If bacteria produce H₂S gas=black precipitatein the butt.
 TSI Slant Results (Color of Slant / Color of Butt)

Yellow I Yellow Yellow I Yellow (cracking in the butt ) Yellow I Yellow (black color in the butt )
Acid I Acid Acid I Acid Acid I Acid

A/ A A/ A A/ A . H2S

Lactose and I or sucrose Lactose and I or sucrose fermenter and Lactose and I or sucrose fermenter and H2S
fermenter bacteria gas producer bacteria gas producer bacteria
 TSI Slant Results (Color of Slant / Color of Butt)

Pink /Yellow Pink / Yellow (cracking in the butt ) Pink /Yellow (black color in the butt )
Alkaline / Acid Alkaline / Acid Alkaline / Acid

K/ A K/ A K/ A . H2S

Non -Lactose and I or sucrose Non -Lactose and I or sucrose Non -Lactose and I or sucrose fermenter and
fermenter bacteria fermenter and gas producer H2S gas producer bacteria
bacteria
 TSI Slant Results (Color of Slant / Color of Butt)

Pink / Pink Pink / Pink (cracking in the butt ) Pink / Pink (black color in the butt )
Alkaline / Alkaline Alkaline / Alkaline Alkaline / Alkaline

K/ K K/ K K/ K . H 2S

Non carbohydrate fermenter, catabolized Gas producer bacteria H2S gas producer bacteria
peptone under aerobic and anaerobic
condition
 TSI Slant Results (Color of Slant / Color of Butt)

Pink / red K/ no change only catabolize peptone under

Alkaline/no change aerobic condition.

If the black color mask the color of butt, the color of the butt is considered yellow.
K/ no change
Why the incubation must be not longer than 18 hours?
Because for example the result is A /A and the bacteria incubate longer than 18
hours, the bacteria ferment lactose and sucrose completely, then start to
degrade peptone which change the pH of the media from acid to alkaline and
the result will appear K I K.

Why there is no A I K?
Because the reaction under aerobic condition is faster than that under anaerobic.
Test #2: IMViC Test
• Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests:

•The 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 species of
bacteria.
 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.

SIM Media : S : for sulfide, I : for Indole, and M: for Motility.

Tryptophane Tryptophanase
Indole + Pyurvic acid + NH3
amino acids

Inoculate from your culture by just single

line inoculation Using sterile cooled needle
incubate at 37°C for 48 hours

Results
SIM Media
 SIM Results
Sulfide test Motility test Indole Test
Black in the No black in Bacteria grow at Bacteria grow at the Add about one 1 ml of kovac's reagent to the
medium the medium the inoculating surface of the media, media
line only or the bacteria grow a If a red ring developed If there is no red ring
way from inoculating at the surface top developed at the
line within 10 minutes surface top within 10
minutes

Sulfide + Sulfide − Non motile Motile Indole + Indole -

 IMViC test
Methyl Red-Voges Proskauer (MR-VP) Tests
Principle: Glucose

Acid None Stable Acid

Stable End Product Acetoin and 2,3 butanediol

Methyl Red Barrit’s reagent

indicator 40% KOH

Red color MR positive VP positive Red color

 Inoculate a loopful of your pure culture
using sterile cooled loop Then divide into 2 vial

Incubate at 37°C for 48 hours.

Add 1 ml Methyl Red Add Barritt's reagent ( alpha

indicator naphthol + 40% KOH)

MR-VP broth

Results
MR : +ve must be VP : -ve
MR: -ve could be VP : +ve or -ve
But impossible to be :MR: +ve VP: +ve

If the whole media converted to the If red ring developed VP

red MR test Positive test Positive
IMViC test:Citrate test
Principle :Often used for the detection of gram-negative enteric bacilli based on the ability of an organism to use
citrate as the source of carbon and energy .

Citrate Pyruvate CO2 + Na + H2O Na2CO3

Alkaline,↑pH

inoculate the slant surface by sterile

cooled loop through streaking
incubate at 37°C for 48 hours.

Results
Citrate +
Simmone’s Citrate slant agar media Citrate -
▪If there is growth
▪and/or the color of the media ▪If there is no
Simmone’s Citrate media Contains growth
converted from green to the blue
Citrate as a carbon source ▪and/or the color
of the media
Bromothymol blue :pH indicator Neutral : Green remains green
 Test # 3: Catalase Test :
Principle : Test for the presence of catalase enzyme that converts hydrogen peroxide (H2O2 ) into water and oxygen gas. When
a drop of peroxide is placed on catalase-producing bacteria, bubbles appear when the oxygen gas is formed.

In Aerobic, Facultative anaerobic, and Aerotolerant anaerobic use O2 as final electron acceptor

Catalase +
▪If there are large amount of bubbles (Strong catalase positive)
▪ If there are small amount of bubbles (Weak catalase positive)

1- Add a loopful from your pure culture Results

into a cleaned slide.
2- Add one drops of 3% H2O2 ( suhstrate)
Catalase -
▪No bubbles
•The End