Increasing importance of clinical microbiology inspired me to work on the clinical diagnosis and microbiology. The presentation will definitely help you to understand the world of microbiology better. https://www.linkedin.com/in/shradheya-r-r-gupta-54492984/

1. Dr. B. Lal Institute of
Biotechnology
(An Exclusive Biotechnology Institute)
Clinical Microbiology
Thursday, January 30, 2020 1
Submitted by:-
Shradheya R.R. Gupta
B.Sc. Biotechnology III Year
Under the guidance of:-
Dr. Yogesh Kumar Singh
Senior Scientist
Dr. B. Lal Clinical Laboratory

2. Introduction To Clinical Microbiology
Clinical microbiology is a branch of medical science
concerned with the prevention, diagnosis and
treatment of infectious diseases.
A clinical microbiologist studies the characteristics
of pathogens, their modes of transmission,
mechanisms of infection and growth.
Thursday, January 30, 2020 2

3. Laboratory Instruments Used
 Autoclave
 pH Meter
 Incubator
 Digital
Balance
Thursday, January 30, 2020 3
Laminar
Air Flow
Hot Plate
Bactec 9050

4. 1. Autoclave
 An autoclave is a device to sterilize articles by subjecting them to
high pressure steam at 121˚C temperature for 15 to 60 minutes
depending on the materials to be sterilized.
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2. pH Meter
A pH meter is an electronic instrument used to measure the pH of a
liquid.
3. Incubator
An incubator is a device used to grow and maintain microbiological
cultures or cell cultures.
4. Digital Balance
 Digital balance is used to measure mass to a very high degree of
precision and accuracy.

5. 5. Laminar Air Flow
 Laminar Air Flow is based on the flow of air current to create uniform
velocity, along parallel lines, which helps to protect the reagents by
providing clean air.
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6. Hot Plate
 A hot plate is a portable self-contained tabletop small appliance that
features one, two or more gas burners or electric heating elements.
7. Bactec 9050
 Bactec 9050 blood culture instrument is designed for the rapid
detection of microorganisms in clinical specimens.

6. Reagent And Media Used
 McFarland Standard
 Blood Agar
 Mac Conkey Agar
 Peptone Water
 Mueller Hinton Agar
 Indole Broth
 Citrate Agar
 Urease Agar
 TSI agar
 MR-VP Broth
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7. 1. McFarland Standard
 McFarland standards are used as a reference to adjust the turbidity of
bacterial suspensions.
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2. Blood Agar
Blood Agar is a enrichment medium used to grow fastidious organisms and to
differentiate bacteria based on their hemolytic properties.
3. Mac Conkey Agar
Mac Conkey agar is a selective and differential culture medium designed to
selectively isolate Gram-negative bacteria.
4. Peptone Water
 The formulation of peptone water permits cultivation of non-fastidious
organisms.
5. Mueller Hinton Agar
It is a non-selective, non-differential medium, which contains starch.

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6. Indole Broth
Production of indole is detected using Kovac’s reagent.
7. Citrate Agar
 Citrate agar to determine the ability of a microorganism to use citrate as its
sole carbon source.
8. Urease Agar
The urease test identifies those organisms that are capable of hydrolyzing
urea to produce ammonia and carbon dioxide.
9. TSI Agar
 TSI agar is used for the differentiation of microorganisms on the basis of
dextrose, lactose, and sucrose fermentation and hydrogen sulfide production.
10. MR-VP Broth
This is to detect the ability of an organism to produce and maintain stable
acid end products from glucose fermentation.

9. Techniques Studied
Screening of bacterial growth using Bactec 9050 in
blood sample.
Gram’s staining of positive blood culture vial.
Sub-culturing on Mac Conkey agar and Blood agar.
Biochemical test for the identification of bacteria.
Antimicrobial susceptibility testing of standard strain
and clinical strain on Mueller-Hinton agar.
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10. 1. Screening of bacterial growth using Bactec 9050 in blood sample.
 Bactec 9050 blood culture instrument is designed for the rapid detection of microorganisms in
clinical specimens.
Thursday, January 30, 2020 10
Each vial was labeled with the appropriate patient information
Vial was loaded in Bactec 9050 culture machine
Vial was left until machine notify the presence of microorganism
Observation:-
If negative- First report will be provided after 48 hours of receiving the samples and second
report will be provided after 5 days.
If positive- A preliminary report of gram stain and critical call out and the final report after
processing & sensitivities are done.

11. 2. Gram’s staining of positive blood culture vial
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Take inoculums on clean slide and make smear
Heat fix
Pour crystal violet and kept it for 1 minute (Wash it with water )
Flood it with iodine for 1 minute (Wash it with water )
Wash it with alcohol for 10 second (Wash it with water )
Pour safrain and kept it for 1 minute
Wash with water
Observe under 100X
Observation:-
Gram's positive cells stains purple and gram's negative cells stains pink.
It is a differential staining method of differentiating bacterial species into two large groups
based on the chemical and physical properties of their cell walls by detecting peptidoglycan

12. 3. Sub-culturing on Mac Conkey agar and Blood agar
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Blood was taken from positive blood culture vial by using syringe
Few drops of blood was pour on both media
Blood was streaked by using loop on both media
Plates were incubate for 24 hours
Plates were observed
A subculture is a new cell or microbiological culture made by transferring some or all cells
from a previous culture to fresh growth medium.

13. Observation:-
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Fig:- 1. Growth on Mac Conkey
Agar
Fig:- 2. Growth on Blood Agar

14. 4. Biochemical test for the identification of bacteria
 It is an approach to know the nutritional and metabolic capabilities of microorganisms. The
methods available include Coagulase, Catalase, Oxidase and IMViC Reactions.
Thursday, January 30, 2020 14
4.1. Observation of Indole:-
Indole positive tube shows a ring of bright pink to red colour in medium and indole negative
tube shows colorless or light yellow color.
4.2. Observation of Citrate:-
Citrate positive tube shows pink color with growth in medium and citrate negative tube
shows no color change.
4.3. Observation of Urease:-
Urease positive tube shows pink color with growth in medium and urease negative tube
shows no color change.

15. Thursday, January 30, 2020 15
4.5. Observation of MR-VP:-
MR positive reaction shows bright red colour, negative reaction shows yellow colour. VP
positive reaction shows eosin pink/red colour, negative reaction shows no colour change.
4.6. Observation of Catalase:-
Catalase positive slide shows bubbles or froth formation, catalase negative slide shows no
bubbles or no froth formation.
4.7. Observation of Cogulase:-
Coagulase positive slide shows coagulation occurs, coagulase negative slide shows no
coagulation.
4.4. Observation of TSI:-
Acid slant / acid butt - dextrose and sucrose fermented or dextrose and lactose fermented or
all the three sugars, dextrose, lactose and sucrose fermented.
Bubbles or cracks present - gas production Black precipitate present - H2S gas production

16. 5. Antimicrobial susceptibility testing of standard strain and clinical
strain on Mueller-Hinton agar
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Colony was dissolved in peptone water
Tubes were incubated for 15-20 minutes
Turbidity was matched with 0.5 Mc Farland
Swabing was done on Mueller-Hinton agar media using swab stick
Plates were incubated and observed after 4 hours
Plates were incubated and observed after 6 hours
Plates were incubated and observed after 8 hours
Plates were incubated and observed after 10 hours
Plates were incubated and observed after 20 hours
Result was analyzed
The purpose of the Kirby-Bauer disk diffusion susceptibility test is to determine the sensitivity
or resistance of pathogenic bacteria to various antimicrobial.

17. Fig:-3. E.coli standard strain on
Mueller-Hinton agar for disk diffusion
method.
Fig:-5. S.aureus standard strain on
Mueller-Hinton agar for disk diffusion
method.
Fig:-4. Pseudomonas aeruginosa standard strain
on Mueller-Hinton agar for disk diffusion
method.
Observation:-
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