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ةيبط هيرهجم ءايحأ Practical Microbiology 2 stage م.م ميظعلادبع ليقع. + راركلا.م.م سيق Lab 12

1. The document describes several microbiology tests used to identify bacteria, including citrate utilization, motility, lactose fermentation, triple sugar iron, and mannitol salt agar tests. 2. The citrate utilization test determines if a bacteria can use citrate as its sole carbon source, while the motility test identifies motile from non-motile bacteria. 3. The triple sugar iron test identifies glucose, lactose and sucrose fermentation and hydrogen sulfide production in gram-negative rods.

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39 views

ةيبط هيرهجم ءايحأ Practical Microbiology 2 stage م.م ميظعلادبع ليقع. + راركلا.م.م سيق Lab 12

1. The document describes several microbiology tests used to identify bacteria, including citrate utilization, motility, lactose fermentation, triple sugar iron, and mannitol salt agar tests. 2. The citrate utilization test determines if a bacteria can use citrate as its sole carbon source, while the motility test identifies motile from non-motile bacteria. 3. The triple sugar iron test identifies glucose, lactose and sucrose fermentation and hydrogen sulfide production in gram-negative rods.

Uploaded by

AAA
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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‫أحياء مجهريه طبية‬ Practical Microbiology 2ndstage

‫الكرار قيس‬.‫م‬.‫ م‬+ ‫عقيل عبدالعظيم‬.‫م‬.‫م‬ Lab 12

3-Citrate Utilization test:


This test is one of several technique used to assist in the identification of
enterobacteria. The test is based on the ability of an organism to use citrate as its
only sole source of carbon and ammonia as its only source of nitrogen.

• Principle:
The test organism is cultured in a medium which contains sodium citrate, an
ammonium salt and the indicator bromothymol blue. Growth in the medium is
shown by turbidity and a change in colour of the indicator from light green to blue,
due to alkaline reaction following citrate utilization.

• Procedure:
Inoculum is streaked over the slant of Simmon’s citrate agar in a tube and
incubated for 24‐48 hrs.
• Result interpretation:
Growth on the slant and change in colour to blue of the medium indicates positive
result.

Positive : Klebsiella pneumonia


Negative : E. coli

4-Motility Test

• Property it tests for: This test is done to help differentiate species of bacteria that
are motile from non‐motile.
• Media and Reagents Used: Motility media contains tryptose, sodium chloride,
agar, and a color indicator.
• How to Perform Test: Stab motility media with inoculating needle.
• Reading Results: If bacteria is motile, there will be growth going out away from
the stab line, and test is positive. If bacteria is not motile, there will only be growth
along the stab line. A colored indicator can be used to make the results easier to see

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‫أحياء مجهريه طبية‬ Practical Microbiology 2ndstage
‫الكرار قيس‬.‫م‬.‫ م‬+ ‫عقيل عبدالعظيم‬.‫م‬.‫م‬ Lab 12

5-Lactose Fermentation
• Property it tests for: This tests for the bacteria’s ability to ferment lactose.
• Media and Reagents: Used: MacConkey agar with crystal violate
How to Perform Test: Inoculate an MacConkey plate using streak plate method and
incubate 24‐48 hours.

• Results :
A positive result is pink after indicator is added (indicating lactose fermentation)
A negative result will have yellow.

9-Triple Sugar Iron Agar (TSI) test

• Principle:

TSI agar is used to determine whether a gram negative rod utilizes glucose and
lactose or sucrose fermentatively and forms hydrogen sulphide(H2S). TSI contains
10 parts lactose: 10 parts sucrose: 1 part glucose and peptone. Phenol red and
ferrous sulphate serves as indicators of acidification and H2S formation,
respectively. The formation of CO2 and H2 is indicated by the presence of bubbles
or cracks in the agar or by separation of the agar from the sides or bottom of the
tube. The production of H2S requires an acidic environment and is indicated by
blackening of the butt of the medium in the tube.

2
‫أحياء مجهريه طبية‬ Practical Microbiology 2ndstage
‫الكرار قيس‬.‫م‬.‫ م‬+ ‫عقيل عبدالعظيم‬.‫م‬.‫م‬ Lab 12

• Method:

1-With a straight inoculating wire, touch the top of a well isolated colony.
2-Inoculate TSI by first stabbing through center of the medium to the bottom of the
tube and then streaking the surface of the agar slant.

3-Leave the cap on loosely and incubate the tube for 18‐24 hours at 35oC in an
incubator.

• Result interpretation:

1.Alkaline slant/no change in the butt (K/NC) = Glucose, lactose and sucrose
non‐utilizer(alkaline slant/alkaline butt) [figure: 1(d)]
2.Alkaline slant/acid butt (K/A) = Glucose fermentation only. [figure: 1(b)]
3.Acid slant/acid butt (A/A), with gas production = Glucose, sucrose, and/or lactose
fermenter. [figure: 1(a)]
4.Alkaline slant/acid butt (K/A), H2S production = Glucose fermentation only.
[figure: 1(c)]
• Quality control:

A/A, with gas: E. coli


K/A, H2S: Salmonella typhi
K/NC: Pseudomonas aeruginosa

3
‫أحياء مجهريه طبية‬ Practical Microbiology 2ndstage
‫الكرار قيس‬.‫م‬.‫ م‬+ ‫عقيل عبدالعظيم‬.‫م‬.‫م‬ Lab 12

10-Mannitol Salt Agar (MSA)

• Property it tests for: This tests for the bacteria’s ability to tolerate % salt
concentration and ferment mannitol. The media is selective because it selects for salt
tolerant bacteria.
• Media and Reagents: MSA media contains nutrient agar, mannitol, 7% sodium
chloride and phenol red indicator.
• How to Perform Test: Inoculate an MSA plate using streak plate method and
incubate 24‐48 hours.
• Reading Results:

-If the organism is tolerant to salt it will grow.


-If the organism is not tolerant to salt it will not grow.
-If the salt tolerant organism can ferment mannitol then there will be yellow zones
around the colonies.
-If the salt tolerant organism cannot ferment mannitol then the media will remain
pink.

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