Methods for STUDY of Micro-

organisms
OBJECTIVES
Microorganisms
Media and Culture
Direct and Indirect Testing
Sterile versus Non-sterile Body Sites
Specimen(Appropriateness,Collection,Trans
port to lab,Inoculation of media,Culture and
isolation,Confirmation,Report)
Microorganisms
Derived from Greek word Microbe
Study of microorganisms called
Microbiology
E.g. Bacteria,Fungi and Protozoa
 Media and Culture
Media: Nutrients (agar, pH indicators, proteins
and carbohydrates) used to grow organism
outside of their natural habitats
Culture: The propagation of microorganisms
using various media
Direct and Indirect Testing
Direct: Demonstration of the presence of a
infectious agent
– Culture
– Microscopy
– Molecular methods such as PCR
Indirect: Demonstration of presence of
antibodies to a particular infectious agent
– Serology
 Sterile versus Non-sterile
Body Sites
Sterile body sites:
– These sites normally do not contain any
bacteria so any bacteria found there are
significant
Example:
Blood
Spinal fluid
Non-sterile body sites:
– These sites are open to the external
environment and normally contain bacteria
Example:
Throat
Feces
Specimen
Appropriateness
Collection
Transport to lab
Inoculation of media
Culture and isolation
Confirmation
Report
Appropriate Specimen
 From relevant body site
– Collected appropriately
Adequate amount
 Appropriate carrying fluid or container
– To maximize isolation of the
organism
– For safety
 Collection
 • No contamination
 • Appropriate equipment
 • Good instructions to patient
Transport to Laboratory
 • Safe packaging
 • Good labeling
 • Temperature
Inoculation of Media
• Use appropriate culture media
– What kind of specimen is it?
– What test did the physician request?
Culturing Microorganisms
• Inoculation
– Viruses
• Cell culture
– Bacteria, fungi
• Enrichment
• Differential
• Selective
Cell culture
 is the process by which cells are grown
under controlled conditions
Culture media
• Used to grow bacteria
• Can be used to:
– Enrich the numbers of bacteria
– Select for certain bacteria and
suppress others
– Differentiate among different kinds
of
bacteria
Microbiological Culture Media
 Enrichment Media
• Bacterial growth means an increase in
numbers of individual cells
• Enrichment media allows microorganisms
increase their numbers
• Contains nutrients that the organisms need in
order to reproduce

Bacterial colonies
growing
in a petri dish
containing
nutrients.
Selective Media
 • Media that suppresses some unwanted organisms while
allowing others to grow
 • Used for isolation of pathogens from nonsterile sites
– Feces
– Water
– Food
 • Examples
– Bile salts suppress Gram positive organisms (MacConkey agar)
– Antibiotics in chocolate agar suppress normal flora in
Martin-Lewis media, selective for N. gonorrhoeae
MacConkey agar Martin-Lewis agar
 Differential Media
• Allows organisms with different
characteristics to be differentiated from each
other
• Most media can be both selective and
differential
Isolation of Individual
Bacteria
• Specimen is “streaked”, using a sterile
loop, onto solid media.
• The agar plates (media) are incubated
at appropriate temperature and
atmosphere
– Often at 35º C.
– Often at 5% CO2
– Usually first examined after 24
hours
“Streaking a Plate”
 Procedure
1.  Flame the loop and wire and streak a loopful of
broth as at A in the diagram. 
  2.  Reflame the loop and cool it. 
  3.  Streak as at B to spread the original inoculum
over more of the agar. 
  4.  Reflame the loop and cool it. 
  5.  Streak as at C. 
  6.  Reflame the loop and cool it. 
  7.  Streak as at D. 
  8.  Label the plate and incubate it inverted. 
  
 Growth of Colonies
• Bacterial Colony
– Result of one bacterium being isolated from
others during “streaking procedure”
– That bacterium grows in numbers exponentially
– Many bacteria have a generation time of 20
minutes
– 272 organisms in one colony after 24 hours!
Classical bacterial identification can only be performed on pure
cultures of bacteria (ideally, all descendants from one bacterial
cell).
 Colony “Picking”
• Sterile needle or loop is touched to surface of
colony and transferred to fresh, sterile media
• Incubation for another 24 hours
Colonies of Bacteria in Pure
Culture
Identification
• Now we have a pure culture of bacteria
• Testing is now done to confirm the
identification of the bacteria culture
– Stains
 Gram Stain
• Classical bacterial
stain
• Differentiates
between two
different groups of
bacteria.
Steps of Gram staining
1. Flood the heat fixed
smear with Crystal
Violet for one minute.
2. Add Iodine solution for
three minute.
3. Decolorize with
Alcohol about thirty
seconds.
4. Add counter stain with
Safranin for one to two
minute.
Ziehl-Neelsen stain
Described by two German doctor;
Franz Ziehl & bacteriologist and 
Friedrich Neelsen
Also known as Acid fast staining
Almost used for Mycobacterium species
 Procedure of ZN stain
E.g. Mycobacterium Tuberculosis
There are eight steps
STEP 1: Flame slides to heat fix
 STEP 2:
Flood the entire slide with Carbol Fuchsin
Note: Ensure enough stain is added to keep the
slides covered throughout the entire staining
step.
 STEP 3:
Using a Bunsen burner, heat the slides slowly
until they are steaming.  Maintain steaming for 5
minutes by using low or intermittent heat (i.e. by
occasionally   passing the flame from the Bunsen
burner over the slides)
Caution: Using too much flame or heat can cause
the slide to break.
 STEP 4:
Rinse the slide with water.
 STEP 5:
 Flood the slide with 3% acid-alcohol and allow to
decolorize for 5 minutes.
Throughout the 5 minutes, continue to flood
the slides with 3% acid-alcohol until the slides are clear
of stain visible to the naked eye. 
To the right are examples of slides insufficiently and
sufficiently flooded with 3% acid-alcohol.
 STEP 6:
Rinse the slide thoroughly with water and then
drain any excess from the slides.
 STEP 7:
Flood the slide with the counter stain,
 Methylene Blue.  Keep the counter stain on the
slides for 1 minute.
 STEP 8:
Rinse the slide thoroughly with water.
 In last :
If all steps are performed correctly you should
have a slide that looks like this.
The Acid Fast Bacilli have rod shape with pink
color.
Report
 • Final report goes to physician
 • The validity of this report is dependent upon:
– Appropriateness of specimen
– Proper collection and adequacy of specimen
– Appropriate transport to lab
– Use of media of known quality
– Culture and isolation by knowledgeable personnel
using equipment known to be operating correctly
– Confirmation by tests of known quality
– Results interpreted and reported by professional
staff
– No transcription or computer errors
Conclusion
Precautions should be taken in every wink
of test..