Bacteria identification from given sample skills: Organism isolation and

Bacteria Identification from Given Sample Skills: Organism Isolation and Antimicrobial Sensitivity Testing

Question for bacterial identification

For bacteria identification of organisms, you can ask if you have any queries related to a given question.

A broth culture is given to you to isolate and identify the organism and also produce its antibiotic sensitivity profile by Saturday. Please do not proceed to another experiment before showing the result of your experiment to your teacher.

Time of inoculation – 1: 00 pm
Type of broth – BHI
Incubation – aerobically
Temperature –?
Isolation from where?
Mixed /single -?

Experimentation Day 1 (Year -month -day i.e. Thursday) for bacterial identification

I was given a broth Labelled as ‘ k’ at 1:00 pm in a broth.
I incubate the broth at 37 ºC for 4 hours aerobically to obtain the growth of the given organ.
Both media BHI (Brain Heart Infusion ) broth.
No added factors/ingredients

After 4 hours (5:00 pm)

Broth shows

Uniform turbidity
No granular formation
No pellicle formation
No pigment formation seen
No gas formation

Gram stain for bacteria identification

Performed on broth with
Positive Control – Staphylococcus accreus ATCC25923
Negative control – Escherichia Coli ATCC 25922

Result of Gram’s stain

Negative control shows – Gram-negative bacilli
Positive control shows -Gram-positive Cocci
Test organism: Gram-negative bacilli
size 1-3 µm x 0.4 – 0.7 µm
Rod-shaped, single, straight rounded ends with parallel side
Arranged singly
No pleomorphic forms seen
No evidence of spore
No evidence of capsule
No mixture pure form seen

Hanging drop preparation for bacteria identification

It shows actively motile organisms.

Possible Pathogens organisms

Escherichia
Salmonella
Proteus
Providencia
Morganella
Pseudomonas
Enterobacter
Burkholderia
Stenotrophomonas
Vibrio
Aeromonas
Plesiomonas
Serratia
Moraxella
Haemophilus
Hafnia

Further Processing

Further, it was processed for isolation and indention; inoculation of broth on

MacConkey agar
Blood Agar
Chocolate
Inoculation By streaking method and plates were incubated at 37 °C aerobically overnight at 5 pm.

Day 2 for bacterial identification

Colony characteristics were studied at 11:00 am from all three plates in transmitted bright light which is helpful for bacteria identification to some extent.

Gram Staining from colonies of three plates

Nutrient Agar : Gram-negative bacilli , 1-3 x 0.4 -0.7 µm
MacConkey agar – Gram-Negative bacilli
Blood Agar – Gram-Negative Bacilli

All three media show similar types of colonies/bacteria in Gram’s stain

Biochemical Tests for bacteria identification

Catalase test for bacteria identification

Positive control – Staphylococcus auress ATCC 25923
Negative control –Enterococcus faecalis ATCC 29912
Test organism – Shows bubbling when adding 3 % H2O2 over colonies

Oxidase Test (Kovac’s Method ) for bacteria identification

Positive control – Pseudomonas aeruginosa ATCC27853
Negative control – Escherichia coli ATCC 25922

Result

Positive control – Deep violet / purple color
Negative control – No color changes

Test Organism – Deep purple/ violet color

Interpretation

Oxidase positive Organism

In Out
Pseudomonas Escherichia
Aeromonas Salmonella
Plesiomonas Citrobacter
Burkholderia Proteus
Providencia
Morganella
Serratia

Identification

Media required
Hgh and Leifson of media
Peptone water broth
TSI
SIM
Citrate
Urea
Decarboxylase media
MHA

I have passed 3-5 well-isolated colonies from nutrient agar plate to peptone water and incubated at 37 ºC for 2-6 hours for 4-6 m keeping the organisms in log phase for AST.
Also, I have passed a single isolated colony from nutrient agar into 4-6 ml peptone water and incubated it at 37 ºC for 2 -6 hours for getting the organism in the log phase for biochemical tests.

After 4 hours of incubation of peptone water, the broth was compared to 0.5 MacFarland Standard that shows 1.5 x 10^8 CFU/ml( 10^6-10^8 CFU/ml) bacterial cells.

AST is performed on the bacterial isolated by Kirby- Bauer disc diffusion method on MHA.

Purity plate

There is a need for ATCC plates of common organisms like

Staphylococcus aureus ATCC 25923

Enterococcus faecalis ATCC 29912

Pseudomonas aeruginosa ATCC27853

Escherichia coli ATCC 25922

Before inoculation both for biochemical tests and AST

After inoculation both for biochemical and AST

Day 3 for bacterial identification

Oxidative Fermentation test (Hugh and Leifson method )

Pseudomonas aeruginasa ATCC 27853
Open tube (Aerobic ) – Yellow
Overlayer paraffin (Anaerobic ) – Blue-green

Fermentative control
Escherichia coli ATCC 25933
Open tube (Aerobic ) – yellow
Overlayer paraffin ( Anaerobic ) – yellow

oxidizer / Non fermenter reaction
Alkaligens faecalis
Open tube (Aerobic) – green
Overlayed Paraffin ( Anaerobic) – Green

Result

Open / Aerobic tube – yellow
paraffin overlayed – green/non change
organism is oxidative

Triple Sugar Iron Agar
A/A H2S – = Escherichia coli ATCC 25922
K/A H2S + , GAS – =Salmonella enterica serotype Typhi
K/A H2S – GAS + = Salmonella enterica serotype Paratyphi A
k/NC H2S- GAS – = Pseudomonas aeruginosa ATCC 278553

Result: K/NC H2S – Gas –

Sulfide indole motility (SIM) test for bacteria identification

Control
Organisms Indole Motility Sulfide
E. coli + + –
Kleb. pneumoniae – – –
Proteus mirabilis – + +
Test organism – + –

Result = Indole- H2S – Modility +

Citrate utilization test for bacteria identification

Positive control – Klebsiella pneumoniae ATCC 13883

Bluish Color
Negative control – Escherichia coli ATCC 25922
Greenish ( no color change)

Test Organism – Bluish color
Result –Citrate utilization test positive

Decarboxylase Test for bacteria identification

Positive control
lysine – Klebsiella pneumoniae ATCC 13883
ornithine – Enterobacter cloacae ATCC 23355
Arginine – Enterobacter cloacae ATCC 23355

Negative control
Lysine – Enterobacter clocae ATCC 23355
ornithine – Klebsiella pneumoniae ATCC 13883
Arginine – Klebsiella pneumoniae ATCC 13883

Test organism
Lysine = negative
Arginine = positive
Ornithine = negative

In Out

Pseudomonas Burkholderia

Pseudomonas aeruginosa isolated.

Pigment: pyocyanin (yellow-green)

Growth at 42°C

Nitrite reduction test positive

Gelatin liquefaction test positive

Antibiotic Susceptibility Testing

Kirby-Bauer Disc diffusion method

Further Processing

Epidemiological markers
Pyocin typing
Bacteriophage typing
Serotyping
Restriction fragment length polymorphism (RFLP)
Ribotyping

G+C context = 50 -70 %

Intrinsic resistance

Ampicillin
Cotrimoxazole
Chloramphenicol
Tetracyclin

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