Basic Microbiology Procedures

MICROBIOLOGY & PARASITOLOGY

1. Basic Microscopy

Refer to previous discussion

2. Handwashing

Refer to previous discussion

3. Staining

3-1 Negative Staining

3-2 Smear Preparation
3-3 Simple Staining
Negative staining- employs the use of an acidic stain
and, due to repulsion between the negative
charges of the stain and the bacterial surface, the
dye will not penetrate the cell. In negative staining,
the results yield a clear cell with a dark background.

Simple staining involves directly staining the bacterial

cell with a positively charged dye in order to see
bacterial detail, in contrast to negative staining
where the bacteria remain unstained against a dark
background.
Negative staining:
4. Gram Staining

4-1 Gram staining is a method of differentiating

species of bacteria into two large groups-
Gram positive or Gramnegative- based on the
chemical and physical properties of their cell
walls
Gram Staining
4-2 Acid fast staining:
Is a special bacteriological stain used to identify acid
fast microorganisms such as mycobacteria
species. It can also be used to stain few other
bacteria like Nocardia.
It is also known as Ziehl-Neelsen staining and it uses
the ff:
Carbolfuschin, acid alcohol, methylene blue
-Acid fast bacilli will appear as bright red after
staining
Gene Xpert:
5. Isolation of Bacterial
Colony
5-1 Spread Plate Technique
5-2 Streak Plate Technique
5-3 Streak Plate Technique
5-4 Preparing a Differential Media
5-5 Pour Plate Technique
 Spread plate technique- is an easy and direct
way.
 Isolated, pure colonies can also be obtained by
the streak plate technique, the key principle of
which is that by streaking, a dilution gradient is
established on the surface of the plate as cells
are deposited on the agar surface.
 Pour plate technique- will also yield isolated
colonies and has been extensively used with
bacteria and fungi.
 6. Preparation and Sterilization of
Culture

6-1 Preparing a Chemically Defined Medium

6-2 Autoclaving
6-3 Preparing Agar Plates
Sterilization- is the process of rendering a
medium or material free of all forms of life.
Autoclaving- is sterilization of items through
exposure to steam at 121 C and 15 lbs of
pressure for 15 minutes or longer.
Dry heat sterilization
Bacteriological filter
Ultraviolet irradiation
Sterilization with ethylene oxide gas
 7. Transfer and Isolation Techniques and
Maintenance of Pure Culture

7-1 Aseptic Technique

7-2 Transferring Culture
7-3 Isolation of Pure Culture and their
Maintenance
 Aseptic vs sterile

  aseptic refers to the collection of practices that

are designed to avoid the introduction and
transfer of germs and contaminants during
medical processes.
 sterile means the same thing as aseptic—germ-
free. However, it’s most often applied to
environments, such as an operating room,
and to instruments and materials, such as
scalpels, needles, and gauze
8. Determination of Bacterial Numbers

8-1 Standard Plate Count

This is an indirect measurement of cell density
and reveals information related only to live
bacteria.
Another method is the spectrophotemetric
analysis which is based on turbidity and
indirectly measures all bacteria, dead or alive.
 9. Testing for Bacterial
Motility
9-1 Hanging Drop Slide
-The bacteria appear to be moving erratically.
- The erratic movement is due to Brownian
movement. This results from the random
motion of the water molecules bombarding
the bacteria and causing them to move
 10. Water Testing

10-1 Membrane Filter Technique

10-2 Total Coliform Test
10-3 Fecal Coliform Test
Total coliform test can be determined by a
statistical estimation called most probable
number test (MPN)
- It involves a multiple series of Durham
fermentation tubes and is divided into three
parts: presumptive, confirmed and completed
tests
An alternative to MPN procedure is the
presence- absence (P-A) test which is based
on the assumption that no coliforms should
be present in 100 mL of drinking water.
Membrane filter technique- involves filtering of
known volume of water through a special
sterile filter.
 11. Food Sanitation

11-1 Bacterial Count in Food Products

11-2 Coliform Analysis in Milk Production
11-3 Methylene Blue Reductase Test for Milk Products

Pasteurization is a means of processing raw milk,

before it is distributed, to assure that it is relatively
free of bacteria and safe for human consumption. It
is a heat process gentle enough to preserve the
physical and nutrient properties of milk, but
sufficient to destroy pathogenic microorganisms.
Methylene Blue Reductase
Test for Milk Products
 12. Blood Typing

12-1 Hemagglutination microscopic-slide tests:

Hemaagglutination- when agglutination

reactions involve the clumping of red cells
This appear due to the antibodies to cross
link red blood cells by binding to surface
antigen
 13. Isolation of Normal
Flora
13-1 Throat Culture
13-2 Skin Culture
13-3 Rectal Culture
13-4 Biochemical Testing

The microorganisms that constitute the normal flora

of the human body are usually harmless, although
some are potential pathogens or opportunists.
These microorganisms may cause disease under
certain circumstances.
E. Coli and E. aerogenes- most common rectal
isolates which can be distinguished by
biochemical tests.
• Select two different colonies from your EMB
plate and inoculate the following media with
each, respectively to:
TSI , LIA, SIM, Simmon Citrate, MR-VP Broth
Biochemical Tests
 1. TSI: (Triple Sugar Iron)
a. Identify your rectal bacteria from the ff.
biochemical test reactions:
i. Acid slant and acid butt with gas ( A/Ag)
ii. Acid slant/ Acid butt (A/A)
iii. Acid slant and acid but with H2S ( A/A with
H2S)
iv. Alkaline slant and alkaline butt ( K/K)
v. Alkaline slant and neutral butt (K/N)
 b. Interpretation:
i. For acid production the medium will turn
yellow
ii. For gas production, the medium will be
broken up
iii. For an alkaline reaction, the medium will be
pinkish
iv. For H2S production, the medium in the butt
will turn black
 2. LIA
a. Observe the LIA tube for the following
reactions:
i. Alkaline slant and alkaline or neutral butt
( K/K or K/N)
ii. Alkaline slant and acid butt (K/A)
iii. Red slant and acid but (R/A)
 b. Interpretation:
i. K/K or K/N lysine –decarboxylase positive
ii. K/A lysine-decarboxylase negative
iii. R/A  lysine-deaminase positive
3. SIM: (sulfide/indole/ motility)
a. Observe the SIM tube for motility and H2S
production.
 Motile cultures will have growth diffusing from the
stab line while nonmotile ones will not.
b. H2S production is indicated by blackening along the
line og growth
c. Add 2-4 drops of Kovac’s reagent to the surface,
shake gently, and allow to stand for few minutes
d. Indole production in indicated by a red color.
4. Simon Citrate- if the citrate slant turns blue,
this indicates citrate utilization and is a
positive test.
5. MR-VP broth
14. Chemicals and Bacteria

14-1 Disinfection
14-2 Use of Antimicrobial Agents

Kirby Bauer- this method employ antibiotics

impregnated to paper disks and then placed on a
seeded Mueller- Hinton agar plate using a
mechanical dispenser.
The inhibition zone that is produced will indicate the
susceptibility or resistance of a bacterium to the
antibiotics
15. Blood Test for Parasites

15-1 Capillary Blood Obtained by Fingerprick

15-2 Thick smear preparation
15-3 Thin smear preparation
Capillary prick
 16. Stool Specimen for
Parasites
16-1 Stool specimen collection
16-2 Formalin-ethyl acetate sedimentation
concentration
Stool specimen collection
17. Stool Specimen Microscopy for Parasites

17-1 Calibration of Microscopes Using an Ocular

Micrometer
17-2 Wet Mount Preparation
18. Other Specimen for
Parasites
18-1 Sputum Specimen Preparation
18-2 Sputum Staining
18-3 Cellulose Tape or Swube Test Procedure
18-3 Urine Specimen Preparation
Tissue samples, including biopsies, surgical or
necroscopy specimens, collected for possible
detection of parasites.
Sputum Specimen Preparation
 Cellulose Tape or Swube Test
Procedure

The adhesive part of the

swube tube or tape is
applied to the perianal
area first thing in the
morning.
For detection of Enterobius vermicularis eggs
(pinworms)