BIOCHEMICAL TEST | Bacterial Identification Technique | Microbiology | Vivek Srinivas |#Bacteriology

Summary

This video presentation delves into various biochemical tests for bacterial identification, essential for definitive diagnosis beyond clinical signs. It covers direct microscopic examination, culturing on different media, and a range of biochemical tests including catalase, oxidase, indole, methyl red, Vogus Proskauer, citrate utilization, urease, sugar fermentation, hydrogen sulfide production, and the Triple Sugar Iron (TSI) test. The script also mentions advanced techniques like PCR, DNA sequencing, and phage typing, concluding with the gelatin liquefaction test, providing a comprehensive guide to bacterial identification in laboratories.

Takeaways

• 🔬 The definitive identification of bacteria requires laboratory examination beyond clinical signs and symptoms.
• 📝 Direct microscopic examination and bacterial staining techniques like Gram, acid-fast, negative, spore staining, and motility tests are initial steps in bacterial identification.
• 🌱 Culturing bacteria on different types of media such as differential, selective, enriched, and enrichment broth is essential for further analysis.
• 🧪 Biochemical tests are crucial for identifying the genus and species of bacteria by assessing their enzymatic properties and substrate utilization.
• 🌡 The catalase test differentiates bacteria based on their ability to decompose hydrogen peroxide, producing oxygen bubbles for positive reactions.
• 🟣 The oxidase test detects the presence of the oxidase enzyme, which turns a specific reagent purple in the presence of bacteria.
• 🔴 The indole test identifies bacteria that produce indole as a by-product when metabolizing tryptophan, indicated by a red color change with Kovax reagent.
• 🍬 The methyl red test measures the acidic by-product of glucose fermentation, which changes the indicator to red in a positive test.
• 🍇 The Voges-Proskauer (VP) test detects the production of acetoin from glucose fermentation, resulting in a red color complex with specific reagents.
• 🍋 The citrate utilization test identifies alkaline pH changes due to citrate metabolism, indicated by a color change to Prussian blue.
• 🔄 The IMViC tests (Indole, Methyl Red, Voges-Proskauer, and Citrate) are a set of biochemical tests used to differentiate members of the Enterobacteriaceae family.
• 🧫 The urease test detects bacteria that hydrolyze urea into ammonia and carbon dioxide, causing an alkaline pH shift indicated by a color change to pink.
• 🍯 The sugar fermentation test determines a bacteria's ability to ferment various sugars, producing acid and possibly gas, which can be observed through color change and bubble formation.
• ⚫ The hydrogen sulfide production test detects the formation of black precipitate due to the reaction between hydrogen sulfide and iron salts.
• 🌊 The triple sugar iron (TSI) test evaluates sugar fermentation and hydrogen sulfide production using a medium with three different sugars and an iron salt indicator.
• 🧊 The gelatin liquefaction test detects bacteria that produce gelatinase, an enzyme that breaks down gelatin even at low temperatures, maintaining a liquefied state.

Q & A

What is the primary purpose of biochemical tests in identifying bacteria?

The primary purpose of biochemical tests is to provide definitive identification of bacteria, which is necessary beyond just clinical signs and symptoms, to identify the causative bacterial agent for infection.

Why is laboratory examination essential for bacterial identification?

Laboratory examination is essential because it allows for the collection of correct samples and the application of various tests, such as microscopic examination, culturing, biochemical tests, serological tests, molecular techniques, and phage typing, which are necessary for definitive bacterial identification.

What is the principle behind the catalase test for bacterial identification?

The principle behind the catalase test is that certain bacteria produce the enzyme catalase, which can decompose hydrogen peroxide into water and oxygen. The release of oxygen is observed as bubbles or effervescence, indicating a positive test.

How is the oxidase test performed and what does it indicate?

The oxidase test is performed by placing a bacterial colony on an oxidase reagent-impregnated disc. If a purple color develops, it indicates the presence of the oxidase enzyme in the bacteria, suggesting a positive test.

What is the purpose of the indole production test and how is it conducted?

The indole production test is used to detect the presence of the enzyme tryptophanase, which converts tryptophan into indole. The test is conducted by inoculating a bacterial colony into a broth containing tryptophan and adding Kovax reagent; a red color development indicates a positive test due to indole production.

What does the methyl red test indicate about bacterial metabolism?

The methyl red test indicates whether certain bacteria ferment glucose to produce pyruvic acid, which lowers the pH to acidic levels. The addition of methyl red indicator to the broth changes the color to red in acidic conditions, indicating a positive test.

What is the significance of the Voges-Proskauer (VP) test in bacterial identification?

The VP test is significant as it detects the production of acetylmethylcarbinol (diacetyl), a neutral byproduct of glucose metabolism in certain bacteria. A positive test shows a red color change when specific reagents are added, indicating the presence of this byproduct.

How does the citrate utilization test help in bacterial identification?

The citrate utilization test helps in identifying bacteria that can utilize sodium citrate to produce carbonate and bicarbonate, which raises the pH to alkaline levels. A color change to prussian blue in the presence of bromothymol blue indicator indicates a positive test.

What is the principle of the urease test and what does a positive result indicate?

The principle of the urease test is based on the ability of certain bacteria to produce the enzyme urease, which hydrolyzes urea into ammonia and carbon dioxide, increasing the pH to alkaline levels. A positive result is indicated by a color change to pink due to the presence of phenol red indicator.

What information can be gathered from the sugar fermentation test?

The sugar fermentation test provides information about the ability of bacteria to ferment specific sugars, producing acid as a byproduct and changing the pH to acidic levels. This can help differentiate between bacterial species based on their metabolic capabilities.

How does the triple sugar iron (TSI) test differentiate between different bacterial species?

The TSI test differentiates bacterial species by observing their fermentation patterns of three different sugars (glucose, sucrose, and lactose) and their ability to produce hydrogen sulfide. Different reactions, such as color changes and gas production, indicate the metabolic characteristics of the bacteria.

What does the gelatin liquefaction test measure and what is its significance?

The gelatin liquefaction test measures the ability of certain bacteria to produce the enzyme gelatinase, which breaks down gelatin into amino acids, even at low temperatures. A positive test, where the gelatin remains liquefied at 4 degrees Celsius, indicates the presence of this enzyme.

Outlines

00:00

🔬 Bacterial Identification Through Biochemical Testing

This paragraph introduces the necessity of biochemical testing for definitive bacterial identification beyond clinical signs and symptoms. It outlines various laboratory methods, including direct microscopic examination using different staining techniques, culturing on different types of media, biochemical tests, serological tests, molecular techniques like PCR and DNA sequencing, and phage typing. The paragraph emphasizes that without biochemical testing, complete identification of bacteria, such as distinguishing between E. coli, Klebsiella, and Citrobacter, is not feasible.

05:00

🧪 Biochemical Tests for Bacterial Identification

This section delves into specific biochemical tests used for bacterial identification, starting with the catalase test, which detects the enzyme catalase that breaks down hydrogen peroxide. It then covers the oxidase test, which identifies bacteria that produce the oxidase enzyme, turning a reagent purple. The indole production test is explained, which detects bacteria that produce indole from tryptophan, changing the Kovax reagent red. The paragraph also describes the methyl red test for glucose fermentation and the Vogus Proskauer test, which detects the production of acetylmethyl carbonyl from glucose fermentation, turning a mixture red.

10:02

🌿 Citrate Utilization and Other Biochemical Tests

The paragraph discusses the citrate utilization test, which identifies bacteria that can change pH to alkaline by utilizing sodium citrate. It also explains the IMViC reactions (Indole, Methyl Red, Vogus Proskauer, and Citrate) used for identifying Enterobacteriaceae family members. The urease test is described, which detects bacteria that produce ammonia and carbon dioxide from urea, changing pH to alkaline. The sugar fermentation test is introduced, which determines a bacteria's ability to ferment different sugars, indicated by a color change and possible gas production.

15:03

🍬 Sugar Fermentation and Hydrogen Sulfide Production Tests

This paragraph focuses on the sugar fermentation test, explaining how bacteria ferment various sugars and produce acids or gases, detected by a color change or bubble formation. It also introduces the hydrogen sulfide production test, which identifies bacteria that produce hydrogen sulfide, forming a black precipitate with iron salts. The Triple Sugar Iron (TSI) test is detailed, a slant test with three sugars and ferrous sulfate to detect sugar fermentation and hydrogen sulfide production, with different reactions observed for E. coli and Salmonella.

20:04

🧊 Gelatin Liquefaction and Summary of Biochemical Tests

The final paragraph describes the gelatin liquefaction test, which detects bacteria that produce gelatinase, an enzyme that breaks down gelatin even at low temperatures. It summarizes the various biochemical tests available for bacterial identification, including their principles and procedures. The paragraph concludes with an invitation to stay tuned for the next video on antimicrobial susceptibility testing and provides a downloadable link to a schematic illustration of the biochemical tests in the video description.

Keywords

💡Biochemical test

Biochemical tests are laboratory procedures used to identify bacteria based on their metabolic activities and the by-products they produce. In the video, these tests are crucial for definitive bacterial identification beyond what clinical signs and symptoms can provide. For example, the script mentions various biochemical tests such as the catalase test and the oxidase test, which are used to differentiate bacterial species based on their enzymatic reactions.

💡Bacterial staining techniques

Bacterial staining techniques, such as gram staining, acid-fast staining, and negative staining, are methods used to color bacteria for microscopic examination. The script explains that these techniques help in the preliminary identification of bacteria by their morphology and staining properties, which is essential before conducting more specific biochemical tests.

💡Culturing

Culturing refers to the process of growing bacteria in a controlled environment on different types of media. The script discusses how culturing on differential, selective, and enriched media helps in the isolation and identification of bacteria. For instance, the script mentions that bacteria producing pink colonies on MacConkey's agar can be further identified using biochemical tests.

💡Gram-negative bacilli

Gram-negative bacilli are a group of bacteria that do not retain the crystal violet dye in the gram staining procedure. The script uses this term to illustrate the outcome of the gram staining test, which is a critical step in distinguishing between different types of bacteria, such as E. coli, Klebsiella, or Citrobacter.

💡Enzyme activity

Enzyme activity is central to many biochemical tests, as it indicates the presence of specific enzymes that bacteria produce. The script describes how the presence or absence of enzymes like catalase and oxidase can help identify certain bacteria. For example, the catalase test results in the production of bubbles if the enzyme is present, as seen with Staphylococcus bacteria.

💡Sugar fermentation

Sugar fermentation is a metabolic process where bacteria convert sugars into acids, gases, or other by-products. The script explains how different bacteria ferment various sugars, leading to changes in pH indicators and the production of gases, which is used to identify the bacteria in tests like the sugar fermentation test and the Triple Sugar Iron (TSI) test.

💡pH indicator

A pH indicator is a substance that changes color in response to the acidity or alkalinity of a solution. In the script, pH indicators like phenol red and bromothymol blue are used in tests such as the urease test and the citrate utilization test to visually indicate the metabolic activity of bacteria by the color change they cause.

💡Serological test

Serological tests detect the presence of antibodies or antigens in a sample and are used to identify bacteria. The script mentions tests like slide agglutination, tube agglutination, and microscopic agglutination tests, which are based on the principle of antigen-antibody reactions to diagnose bacterial infections.

💡Molecular techniques

Molecular techniques, such as PCR (Polymerase Chain Reaction) and DNA sequencing, are advanced methods used for bacterial identification. The script briefly mentions these techniques as part of the laboratory strategies for definitive bacterial identification, providing a more precise and accurate identification than traditional methods.

💡Phage typing

Phage typing is a method of bacterial identification that uses bacteriophages, or viruses that infect bacteria, to differentiate between strains. The script describes this as the last step in the series of tests for bacterial identification, showcasing the diversity of methods available to microbiologists.

💡IMViC reactions

IMViC reactions stand for Indole, Methyl Red, Voges-Proskauer, and Citrate tests, which are a set of four biochemical tests used to identify members of the Enterobacteriaceae family. The script uses the results of these tests to differentiate between E. coli, Salmonella, and Klebsiella, demonstrating their utility in bacterial classification.

Highlights

Laboratory examination is essential for definitive bacterial identification beyond clinical signs and symptoms.

Different bacterial staining techniques like gram staining, acid-fast staining, and negative staining are used for microscopic examination.

Bacterial culture media types include differential, selective, enriched media, and enrichment broth for bacterial growth.

Biochemical tests are crucial for identifying bacterial genus, species, and sometimes subspecies.

The catalase test detects the presence of the catalase enzyme, which breaks down hydrogen peroxide.

The oxidase test identifies bacteria that produce the oxidase enzyme, changing a reagent to a purple color complex.

Indole production test detects bacteria that produce indole from tryptophan, forming a red ring with Kovax reagent.

Methyl red test indicates glucose fermentation by detecting a change to an acidic pH and a red color.

Voges-Proskauer test detects the production of diacetyl from glucose fermentation, turning a mixture red.

Citrate utilization test identifies bacteria that utilize sodium citrate, changing pH to alkaline and turning bromothymol blue to prussian blue.

IMViC reactions, a set of four biochemical tests, are used for identifying members of the Enterobacteriaceae family.

Urease test detects bacteria that hydrolyze urea to ammonia and carbon dioxide, changing pH to alkaline and turning phenol red to pink.

Sugar fermentation test determines which sugars a bacteria can ferment, producing acid and changing pH to acidic.

Hydrogen sulfide production test detects the production of hydrogen sulfide, forming a black precipitate with iron salts.

Triple sugar iron (TSI) test checks for sugar fermentation and hydrogen sulfide production in a single test.

Gelatin liquefaction test detects bacteria that produce gelatinase, breaking down gelatin even at low temperatures.

A downloadable link for schematic illustrations of different biochemical tests is available in the video description.

Upcoming video presentations will cover antimicrobial susceptibility testing.

Transcripts

[Music]

in this video presentation we will see

about the different biochemical test for

the identification of the bacteria

the diagnosis of the bacterial infection

can be done based on the clinical signs

and the symptoms of the disease but it

gives only the tentative identification

but for the definitive identification

the laboratory examination is necessary

by the laboratory examination we can

identify the causative bacterial agent

for the infection but for that the

correct sample from the patient has to

be collected based on the clinical signs

first the collected sample can be

subjected for the direct microscopic

examination by performing the different

bacterial staining techniques like the

gram staining the acid fast staining the

negative staining the spore staining and

the motility test

next by culturing the bacteria onto the

different types of bacterial culture

media like the differential media the

selective media the enriched media and

the enrichment broth and next by the

biochemical tests this tests will be

detailed in this video presentation

next by the serological test like the

slide agglutination test the tube

agglutination test and the microscopic

agglutination test and next by using

molecular techniques like the pcr

followed by the dna sequencing and

lastly the phage typing that is the

bacterial identification by using the

bacteriophages these are the laboratory

methods and the strategy followed for

the bacterial identification only by the

microscopic examination and by the

culturing technique the complete

identity of the bacteria is not possible

for instance the bacteria producing the

pink colonies on the mcconkey's agar

when subjected to the microscopic

examination will shows the gram-negative

bacilli which may be the e coli the

klebsiella or the citrobacter so the

genus and species level bacterial

identification is achieved by subjecting

the bacterial culture to various

biochemical tests and other techniques

the biochemical test this helps in the

identification of the bacterial genus

and the species sometimes even the

subspecies too this is achieved by

subjecting the bacterial culture to the

various biochemical tests

the various bacteria has its inherent

property to produce the different types

of the enzymes and the substances in

addition the various bacteria also has

its inherent property to ferment or to

utilize the different types of the

sugars and the substrates the yield the

different types of by-products this can

be identified by subjecting the

bacterial culture to the various

biochemical tests

first we see about the catalase test the

principle certain bacteria produce the

enzyme the catalase this enzyme along

with the hydrogen peroxide capable of

decomposing the hydrogen peroxide to the

water and the oxygen the release of the

oxygen is observed as the bubbles or the

effervescence formation this is the

principle behind the catalase test

the procedure take a glass slide

place 0.5 ml of the hydrogen peroxide

over the slide then emulsify the

bacterial colony to the hydrogen

peroxide if there is no bubbles or no

effervescence observed indicates the

test is negative

the example the streptococcus bacterial

organism negative for the catalase test

in contrast if the bacterial colony once

emulsified with the hydrogen peroxide

produce bubbles or effervescence

indicates the test is positive the

example the staphylococcus bacterial

organism positive for the catalase test

this is the procedure for the catalase

test

this is the photograph showing the

positive and the negative test result of

the catalase test

next the oxidase test the principle

certain bacteria produce the enzyme the

oxidase this enzyme along with the

oxidase reagent consisting of one

percent

tetramethylparaphenyldiamine

dihydrochloride capable of turning into

the endophenol this into phenyl is a

purple color complex this is the

principle behind the oxidase test

the procedure take the oxidase disc this

disc is the oxidase reagent impregnated

disc now place the bacterial colony over

the oxidase disc if there is no color

development observed indicates the test

is negative the example the e coli

bacterial organism negative for the

oxidase test in contrast following

addition of the bacterial colony over

the oxidase disc produces the purple

color indicates the test is positive the

example the pseudomonas bacterial

organism positive for the oxidase test

this is the procedure for the oxidase

test

this is the photograph showing the

positive and the negative test result of

the oxidase test

next the indole production test or the

indole ring test the principle

certain bacteria produce the enzyme the

tryptophanase this enzyme in presence of

the tryptophan capable of utilizing the

tryptophan to yield the indole as the

by-product

next this indole by-product once mixed

with the kovax reagent develop a red

color complex this is observed as the

red ring this is also referred as the

indole ring this is the principle behind

the indo-ring test

the procedure this test is performed in

the test tube with the liquid broth

containing the tryptophan now take the

bacterial colony inoculate into this

broth incubate for 24 to 48 hours

following incubation add the covax

reagent along the side the formation of

red ring which indicates the test is

positive due to indole production the

example the e coli bacterial organism

positive for the indole test

in contrast following addition of the

covax reagent if there is no red ring

formation which indicates the test is

negative for the endoproduction the

example the salmonella bacterial

organism negative for the endo test this

is the procedure for the endo test

this is the photograph showing the

positive and the negative test result of

the indole production or the indole ring

test

next the methyl red test also

abbreviated as the mr test the principle

certain bacteria in presence of the

glucose ferment the glucose to yield the

pyruvic acid as the byproduct this

changes the ph 2 acidic from the initial

neutral ph

next this acidic ph along with the

methyl red indicator develops the red

color this is the principle behind the

methyl red test

the procedure this test is performed in

the glucose phosphate broth with the

neutral ph

now take the bacterial colony inoculate

into this broth incubate for 24 to 48

hours following incubation add the

methyl red indicator to it the

development of red color which indicates

the test is positive due to acidic ph

the example the e coli bacterial

organism positive for the mr test

in contrast following addition of the

methyl red indicator if there is no

color change which indicates the test is

negative for the mr test the example the

klebsiella pneumoniae bacterial organism

negative for the mr test this is the

procedure for the methyl red test

this is the photograph showing the

positive and the negative test result of

the methyl red test

next the vogus prosper test also

abbreviated as the vp test the principle

certain bacteria in presence of the

glucose ferment the glucose to the acid

and then subsequently metabolize to

yield a neutral byproduct the acetyl

methyl carbonyl which is an acetoin

compound

next this acidoin compound along with

the 40 potassium hydroxide and the 5

alpha naphthal to form the red color

complex the diacetyl derivative this is

the principle behind the vogus prosper

test

the procedure this test is performed in

the glucose phosphate broth now take the

bacterial colony inoculate into this

broth incubate for 48 hours

following incubation add the 5 alpha

naphthal and the 40 potassium hydroxide

to it the development of the red color

which indicates the test is positive due

to the diacetyl formation the example

the klebsiella pneumoniae bacterial

organism positive for the vp test

in contrast following addition of the 5

alpha naphthal and the 40 potassium

hydroxide if there is no color change

which indicates the test is negative for

the vp test the example the e coli

bacterial organism negative for the vp

test

this is the procedure for the vp test

this is the photograph showing the

positive and the negative test result of

the vp test

in general the vp test is done in

conjugation with the mr test the

bacterial organisms showing the mr test

positive will give the vp test negative

and the bacterial organisms showing the

mr test negative will give the vp test

positive therefore in general these two

test results are vice versa for the

bacteria

next the citrate utilization test

the principle certain bacteria in

presence of the sodium citrate utilize

the citrate the yield the carbonate and

the bicarbonate as the byproduct this

changes the ph to alkaline from the

initial neutral ph this is the principle

behind the citrate utilization test

the procedure this test is performed in

the test tube with the agar slant

prepared with the simon citrate agar

containing the sodium citrate and the

bromothymol blue as the indicator this

bromothymol blue indicator at neutral ph

appears as the deep forest green

now take the bacterial colony inoculate

into this agar slant incubate for 24 to

48 hours

following incubation if the agar slant

turns to the prussian blue which

indicates the test is positive due to

the changes in the ph 2 alkaline because

of the yield of the carbonate and the

bicarbonate as the byproduct this

bromothymol blue indicator at alkaline

ph appears as the prussian blue the

example the salmonella bacterial

organism positive for the citrate test

in contrast following incubation if

there is no color change which indicates

the test is negative for the citrate

utilization test the example the e coli

bacterial organism this is the procedure

for the citrate utilization test

this is the photograph showing the

positive and the negative test result of

the citrate utilization test

the imvik reactions there are the set of

four biochemical tests they are commonly

employed in the identification of the

members of the family anterobacteria ce

bacteria the four biochemical tests are

the indo-ring test the methyl red test

the vogus prosker test and the citrate

utilization test the letter i is only

for the rhyming purpose the e coli

bacteria gives plus plus minus minus for

the invic reactions the salmonella

bacteria gives minus plus minus plus for

the invic reactions the klebsiella

bacteria gives minus minus plus plus for

the invic reactions so immvic reactions

are employed in the identification of

the members of the family enterobacteria

ce bacteria

next the urease test also referred as

the urea hydrolysis test the principle

certain bacteria produce the enzyme the

urease this enzyme in presence of the

urea capable of hydrolyzing the urea the

yield the ammonia and the carbon dioxide

as the byproduct this byproduct changes

the ph 2 alkaline from the initial

neutral ph this is the principle behind

the urease test the procedure this test

is performed in the test tube with the

agar slant containing the urea and the

phenyl red as the indicator this phenol

red indicator at neutral ph appears as

the orange now take the bacterial colony

inoculate into this agar slant incubate

for 24 to 48 hours

following incubation if the agar slant

turns to the pink which indicates the

test is positive due to the changes in

the ph 2 alkaline because of the yield

of the ammonia and the carbon dioxide as

the byproduct this phenol red indicator

at alkaline ph appears as the pink the

example the staphylococcus bacterial

organism positive for the urease test

in contrast following incubation if

there is no color change which indicates

the test is negative for the urease test

the example the e coli bacterial

organism negative for the urease test

this is the procedure for the urease

test

this is the photograph showing the

positive and the negative test result of

the urease test

next the sugar fermentation test

each bacteria has the ability to ferment

certain sugars and not ferment certain

sugars to find out the sugar

fermentation of the particular bacteria

this test is done

the principle the bacteria in presence

of the certain sugar the sugar may be

glucose or sucrose or lactose or maltose

or galactose or mannose or any other

sugars this bacteria in presence of the

certain sugar ferment the yield the acid

as the byproduct this changes the ph 2

acidic from the initial neutral ph

next this acid along with the androte's

indicator changes to the pink color due

to acidic ph

some bacteria may also releases the

gases along with the acids this is

observed as the bubble formation this is

the principle behind the sugar

fermentation test

the procedure this test is performed in

the broth containing sugar with the

durham's tube inside it the sugar may be

glucose or sucrose or lactose or maltose

or galactose or mannose or any other

sugars that can be added to the broth

for instance this broth is added with

the lactose thereby to check the

bacteria for the lactose fermentation

now take the bacterial colony inoculate

into this broth incubate for 24 to 48

hours

following incubation add the andronte's

indicator to it the development of the

pink color indicates the test is

positive for the lactose fermentation

due to the acid production

sometimes the bubble formation may be

observed in the durham's tube indicates

the gas production the example the e

coli bacterial organism positive for the

lactose fermentation

in contrast following addition of the

androtes indicator if there is no color

change which indicates the test is

negative for the lactose fermentation

the example the salmonella bacterial

organism negative for the lactose

fermentation this is the procedure for

the sugar fermentation test

next the hydrogen sulfide production

test

the principle certain bacteria produce

substance like the hydrogen sulfide this

hydrogen sulfide along with the iron

salts like ferrous sulfate and ferric

ammonium salt to form the ferrous

sulfide as the end product

this is observed as the black

precipitate this is the principle behind

the hydrogen sulfide production test

next the triple sugar iron test also

referred as the tsi test the procedure

this test is performed in the test tube

with the agar slant prepared with the

tsi agar containing the three sugars the

glucose the sucrose and the lactose to

check the bacteria for the sugar

fermentation for these three sugars and

also contain the ferrous sulfate salt to

check the bacteria for the hydrogen

sulfide production and contain the

indicator the phenyl red this phenol red

indicator at neutral ph appears as

orange generally the top slanting

portion of the agar is referred as the

slant and the bottom portion of the agar

is referred as the butt

the three sugars present in the tsi agar

are in different concentration such as

the glucose 0.1 percent the sucrose one

percent and the lactose one percent this

is about the tsi agar now take the

bacterial colony inoculate into this

agar slant incubate for 24 to 48 hours

following incubation the agar slant may

turns to this type of reaction this type

of reaction is observed in the e coli

that is the yellow slant due to acidic

ph at the slant portion and the yellow

butt due to acidic ph at the butt

portion

and the bubble cracks and the

displacement of the agar due to the gas

production

since the e coli ferments all the three

sugars present in the media so a large

amount of acids are produced which turns

the phenol red indicator to yellow both

at the butt portion and at the slant

portion this type of reaction is

observed in the e coli

following incubation the agar slant may

turns to this type of reaction this type

of reaction is observed in the

salmonella that is the red slant due to

alkaline ph at the slant portion and the

yellow butt due to acidic ph at the butt

portion and also we can observe the

presence of the black precipitate due to

hydrogen sulfide production remember the

butt portion comparatively contain more

glucose compared to the slant portion

since the salmonella ferments only

glucose present in the media a small

amount of the acid is produced which

turns the phenol red indicator to yellow

only at the butt portion this type of

reaction is observed in the salmonella

these are some of the reactions observed

in the tsi agar starting from the left

the uninoculated dsi agar the middle the

e coli inoculated dsi agar and the right

the salmonella inoculated dsi agar

this is the photograph showing the

reactions observed in the tsi agar the

left the uninoculated dsi agar the

middle the e coli inoculated dsi agar

and the right the salmonella inoculated

dsi agar

therefore the e coli ferments all the

three sugars present in the media and

turns the phenol red indicator to yellow

both the butt portion and the slant

portion but the salmonella ferments only

glucose present in the media and turns

the phenol red indicator to yellow only

the butt portion

next the gelatin liquefaction test also

referred as the gelatin hydrolysis test

in this experiment the gelatin will be

used before that we will see about the

property of the gelatin the gelatin at

high temperature that is at temperature

above 25 degrees celsius it liquefies in

contrast the gelatin at low temperature

that is at temperature below 15 degrees

celsius it solidifies this is the

property of the

gelatin now we will see about the

principle of this test

certain bacteria produce the enzyme the

gelatin is this is the proteolytic

enzyme this enzyme in presence of the

gelatin capable of breaking down the

gelatin into the individual amino acids

as the end product this liquefies the

gelatin and remain liquefied even at

four degrees celsius that is remain

liquefied even at the chilling

temperature this is the principle behind

the gelatin liquefaction test

the procedure this test is performed in

the test tube prepared with the nutrient

gelatin medium now take the bacterial

colony inoculate into this gelatin

medium incubate for the 48 hours

following incubation if the medium

remains liquefied even at the 4 degrees

celsius indicates the test is positive

due to the liquefaction of the gelatin

the example the staphylococcus aureus

bacterial organism positive for the

gelatin liquefaction test

in contrast following incubation if the

medium remains solidified at the 4

degree celsius indicates the test is

negative due to the gelatin is not

hydrolyzed by the bacteria the example

the e coli bacterial organism negative

for the gelatin liquefaction test this

is the procedure for the gelatin

liquefaction test

this is the photograph showing the

positive and the negative test result of

the gelatin liquefaction test

the schematic illustration of the

different biochemical test is available

as the downloadable link in the below

youtube description in next video

presentation we will see about the

antimicrobial susceptibility test stay

tuned to this youtube channel hope the

lecture is informative and useful

thank you