Bacteriology

Department of Microbiology
College of Medicine
University of Baghdad

2015-2016
 Learning Objectives
• Understand why specimen collection is Important in
Microbiology
• List types of specimens for microbiology lab
• List types of differential stains
• Define culture media
• Identify the basic ingredients of culture media
• List types of culture media
• Explain the laboratory identification methods of
bacteria
 Specimen collection
Why specimen collection is Important in Microbiology
• Specimen collection in Microbiology to isolate and
identify the causative agents forms back bone of the
investigative procedures.
• In developing world, lack of awareness and casual
attitude among junior staff hampers the definitive
diagnosis.
• Specific procedures in collecting specimens will certainly
improve the quality of services of Microbiology
Departments
 Preparation Smear
 What is Smear?
A specimen for microscopic study prepared by
spreading the material across the glass slide.
 What is Culture ?
Propagation of microorganisms in a growth media.
 What is Growth media?
An artificial media contains basic requirements
needed for microorganisms' growth.
 We have 2 main forms of growth media :
 Growth media
•Liquid or called (broth) usually put it in test tube.

Sterile Broth

•Solid or called (agar) usually put it in Petri dish.

COLONY
Sterile
Petri
 Staining of Bacteria
Bacteria cells are almost colorless and transparent

A staining technique is often applied to the cells to

color them →
Their shape and size can be easily determined
under the microscope.

6
 Types of staining techniques

Simple staining Differential staining

(use of a single stain) (use of two contrasting stains
separated by a decolorizing agent)

For visualization of
morphological Identificatio Visualization
shape & arrangement. n of structure
Gram Acid fast
stain stain Spore Capsule
stain stain
7
Shapes of Bacteria
 Culture Media
 An artificial media contains basic requirements
needed for microorganisms' growth.

 Used for recognition and identification of

microorganisms.

 Bacteria like any living cell it needs organic and

inorganic material for their live.
 Culture Media
 The media are contained either in
• Sterile test tubes
• Plates (Petri Dishes)
• Flasks or screw capped bottles

 The media sterilized by moist heat.

 Basic Ingredient For Culture Media
Peptone: source of protein.
Meat extract: source for N2
Nacl: for isotonic environment.
H2O
Agar Agar: it provides no nutritional benefits but
used for solidifying purposes, it is obtained from
seaweeds
pH: most pathogenic bacteria needs a PH of 7.2-
7.4, this obtained by addition of NaOH or HCL.
 Types of culture media
A. Based on their consistency
a) Liquid medium
b) Solid medium
c) Semi solid medium
B. Based on the constituents/ ingredients
a) Simple medium
b) Special media
C. Based on Oxygen requirement
a) Aerobic media
b) Anaerobic media
 Types of culture media
Special media
– Enriched media (Blood agar & chocolate agar)
– Enrichment media (Selenite-F-broth)
– Selective media (MacConkeys agar)
– Indicator media (Sugar media)
– Differential media (Blood agar& MacConkeys agar)
– Transport media (Stuarts media)
– Sensitivity media (Muller hinton media)
 Nutrient broth
Nutrient agar Blood agar Muller hinton media Chocolate agar

-Why differential?
Bec. it differentiate
bet. Hemolytic &
non hemolytic bac.

Sterile MacConkeys Agar

Cultured MacConkeys Agar
Blood agar
-Why selective? Because it contains bile Salt &
crystal violet that inhibit g+ve bacteria and let g-ve
bacteria to grow
-Why differential? Because it contains lactose which
could differentiate between g-ve bacteria as two group
lactose fermenter and non fermenter

Transport media
sugar media
 Pure Culture
 It is a single kind of microorganisms growing alone in
a protected environment.
 Used for the preparation of pure cultures from
laboratory specimens containing mixed
microorganisms to easily dx.
 Media and equipment to be used must be sterile. and
 Technique must be aseptic.
 Cultural characteristics

Macroscopic characteristics of a pure culture:

1) Size: measured in m.m.
2) Shape: punctiform, circular, filamentous, irregular, spindle.
3) Elevation: flat, raised, convex, umbonate.
4) Margin: entire, lobate, filamentous.
5) Consistency: dry, mucoid.
6) Surface texture: smooth, rough
7) Color or pigmentation: yellow, green …
8) Optical density: Opaque, translucent, glistening.
9) Odor : bad odor, sweat musty odor.
10) Changes in the inoculated medium (hemolysis, bile precipitation) .
Biochemical tests for the isolated
microorganism
• Detect of enzyme activity: Example
1. Coagulase (clotting factor)
Normal coagulation of plasma
Thrombin
Fibrinogen Fibrin
(soluble ) (insoluble)
Staphylococcus aureus coagulation of plasma*
“Free” staphylocoagulase

Fibrinogen + CRF Fibrin

 Biochemical tests for the isolated
microorganism
 Detect of enzyme activity:
Example
1. Coagulase test

DETECT the ability of S. aureus to

clot blood plasma (fibrinogen 
fibrin)

Slide Tube
method method
 Biochemical tests for the isolated
microorganism
• Detect of enzyme activity: Example
2. Catalase test : Inactivates H2O2.
Antimicrobial activity of microorganism

• Antibiotic sensitivity test

Muller hinton media is used to test antibiotic
sensitivity.
• e.g.
Zone of inhibition (no Antibiotics
growth) disc
 Biochemical tests for the isolated
microorganism
• Sugar fermentation : Example Staphylococcus aureus
ferment the Manitol sugar in Manitol salt agar, this medium
contain: Mannitol +7.5% NaCL + Phenol red (indicator),
pink color turn to yellow because of mannitol fermentation
& acid production, & the salt inhibit most other normal
flora.
 Biochemical tests for the isolated
microorganism
 API system ( API= analytic profile index)
This become popular for rapid identification of
microorganisms. Ex: API 20E for rapid identification of
members of the Enterobacteriaceae and other G-ve
bacteria.
Plastic strips consist of 20 small wells containing
dehydrated media components(consist of 20 tests).
 Serology
• Ex: Quelling reaction (Capsular swelling
test)
Rapid diagnosis test for S.pneumoniae is the Quelling
reaction test: Is a rapid diagnostic test on sputum or
culture.
{S.pneumoniae + specific Ab = capsule swelling (+ve
result) stain with negative stain like India ink}.
Pathogenicity of Microorganisms

• Virulence in mice: when inject M.O in the

mice, the mice will die within 48 hours.
 Today works
Examine slide with oil immersion lens
•