Contents
General objective ......................................................................................................................................... 2
Gram Staining ............................................................................................................................................... 4
MATERIAL USED ........................................................................................................................................ 4
PRINCIPLE OF THE TEST............................................................................................................................. 4
PROCEDURE .............................................................................................................................................. 4
RESULT OF THE TEST ................................................................................................................................. 5
APPLICATION OF THE TEST........................................................................................................................ 5
AFB stain (ziehl-Neelsen staining) ............................................................................................................... 6
MATERIAL USED ........................................................................................................................................ 6
PRINCIPLE OF THE TEST............................................................................................................................. 6
PROCEDURE .............................................................................................................................................. 6
RESULT OF THE TEST ................................................................................................................................. 7
APPLICATION OF THE TEST........................................................................................................................ 7
Wet mount ................................................................................................................................................... 8
MATERIAL USED ........................................................................................................................................ 8
PRINCIPLE OF THE TEST............................................................................................................................. 8
PROCEDURE .............................................................................................................................................. 8
RESULT OF THE TEST ................................................................................................................................. 8
APPLICATION OF THE TEST........................................................................................................................ 8
Cell culture .................................................................................................................................................... 9
MATERIAL USED ........................................................................................................................................ 9
PRINCIPLE OF THE TEST............................................................................................................................. 9
PROCEDURE .............................................................................................................................................. 9
RESULT OF THE TEST ................................................................................................................................. 9
APPLICATION OF THE TEST........................................................................................................................ 9
Reference .................................................................................................................................................... 10
1
General objective
The main objective of this experiment was examination of bacterial specimens to determine their
shape, color, locomotion, and antibiotic resistance by using different techniques.
Mainly we had observed two techniques of examination: direct microscopic examination and
culturing. Direct microscopic examination of specimens often provides the etiological diagnosis
of an infectious process and opportunities to initiate appropriate therapy before the result of
cultures becomes available. This includes staining and wet mount preparation.
1. STAINING: identification of bacteria using colored dyes; it can be gram staining or AFB
stain.
Gram staining is a differential staining procedure used to categorize bacteria as gram positive and
gram negative based on the chemical and physical properties of their cell wall. Gram positive cell
have a thick peptidoglycan in the cell wall that retain the primary stain whereas gram negative cell
has thinner peptidoglycan in the cell wall, so it is easily washed off by alcohol.
Gram staining is almost always the first step in the identification of a bacterial group. While gram
staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be
definitively classified by this technique. This gives rise to gram-variable and gram-indeterminate
groups.
Acid fast bacillus or AFB stain is a differential stain that's used to identity acid fast organisms like
nocardia and mycobacterium.
AFB is a type of bacteria that cause tuberculosis and other chronic infection. Tuberculosis is
commonly called TB, a bacterial infection that affects the lungs.it also affect all body parts,
including the brain, spine, and kidney. The person suffering from TB majority show symptoms of
coughing and sneezing. AFB tests ate usually recommended for people who have symptom of
2
active TB. These tests help to diagnose the presence of AFB bacteria in your sputum. Sputum is
thick mucus that is coughed up in the lungs.it is different from saliva.
2. WET MOUNT PREPARATION: is a procedure in the laboratory to observe motile
organisms.it is commonly used to examine materials collected from vaginal wall of a
female patient.
Culturing simply involves the techniques of growing microorganisms in special media in the
laboratory.it involves making of sterile medium, inoculating, incubating, and examining
microorganisms. By this means, microorganism characteristics such as color, pattern of growth
and appearance can be seen.
3
Gram Staining
MATERIAL USED
➢ Glass slide
➢ Bacterial sample
➢ Gram staining reagent
✓ Gentian violet stain
✓ Mordant or Lugol ‘iodine
✓ Ethanol
✓ Safranin/neutral red
➢ Distilled water
➢ Compound microscope
➢
Glove
➢ Dropper
PRINCIPLE OF THE TEST
Staining is a microscopic technique used to increase visualization of organism. Of which one is
Gram staining that used to differentiate bacteria as gram negative and gram-positive
microorganism. The principle of the test is based on the ability of microorganism to retain color
of the stain used during gram stain reaction. This differential staining is occurring due to the
difference in cell wall composition of gram negative and gram-positive bacteria. Gram positive
bacteria has thick layer of peptidoglycan (the major component of cell wall which composed of
N-acetyl glucosamine and N-acetyl muramic acid). This thick layer of peptidoglycan allows
gram positive bacteria to retain the crystal violet and stain purple and gram-negative bacteria has
thin layer of peptidoglycan, so it doesn’t retain the color of the dye and
stain pink/red.
The color of microorganism is seen under compound microscope. The common arrangement in
the stained smear is either in chain or in cluster. However, it is only be used to describe the
Gram-positive bacteria and there is no significant to use to describe Gram negative bacteria.
PROCEDURE
1. Sample of bacteria was taken.
2. Crystal violet was added and kept for 1 min,
4
3. The crystal violet from the slide was washed with water.
4. Mordant (Lugol’s iodine) was added and kept for 1 min.
5. The slide was washed with water.
6. The decolorizer (acetone/ethyl alcohol) was added then washed with water.
7. A counter stain safranin/neutral red was added and washed with water.
8. Air dry and the slide was examined under compound microscope.
RESULT OF THE TEST
We had two results.
1. Gram positive staphylococci which retain the color of crystal violet and stained purple
and
2. Gram negative bacilli
Fig 1. Gram positive staphylococci
Fig 2. Gram negative bacilli
APPLICATION OF THE TEST
This procedure is used to diagnosis bacterial infection.
5
AFB stain (ziehl-Neelsen staining)
MATERIAL USED
➢ slide
➢ tong
➢ Staining reagent
✓ methylene blue
✓ Alcohol(decolorizer)
✓ carbol fuchsin
➢ water
➢ microscope
PRINCIPLE OF THE TEST
Mycobacterium tuberculosis contain mycolic acid on their cell wall which is a waxy and resistant
to staining with gram stain so we should use acid fast stain. The primary stain or carbol fuchsin
was applied to cells, then we add heat and alcohol or phenol that are used to allow the stain to
penetrate into cell wall of acid-fast microorganisms.
PROCEDURE
1. A specimen smear was taken and fixed on the slide.
2. the slide was placed over a boiling hot water bath.
3. primary stain or carbol fuchsin was added and heated until vapor is observed then the
slide was kept for 5min.
4. slide was rinsed with water until the solution runs clear.
5. acid alcohol decolorizer was added over the slide for 10 to15 sec then kept for 2-3min.
6. The slide was rinsed with water.
7. secondary or counter stain methylene blue was added and kept for 1 min.
8. The slide was rinsed with water.
9. Air dry then examined under microscope.
6
RESULT OF THE TEST
when we examine the specimen under microscope the color of bacteria stay retained the primary
stain color (carbol fuchsin) which means red with blue color background therefore
mycobacterium tuberculosis is acid fast microorganism.
APPLICATION OF THE TEST
AFB stain is the golden standard and most common method to identify different gram variable
bacteria that are difficult to differentiate whether they are gram negative or positive.
To detect M. tuberculosis in specimen sample to diagnose TB in patients.
7
Wet mount
MATERIAL USED
•
Microscope eye piece at 40x resolution /100 x rarely used for detecting motility.
•
Slide/ specimen containing bacteria from agar.
PRINCIPLE OF THE TEST
•
A drop of bacteria suspension is placed on a slide covered with cover slip and
observed under compound microscope or by using oil immersion objective to
increase the specimen translucency.
PROCEDURE
1. a thin slice of sample was collected and placed on clean, dry slide.
2. one drop of water was added over the sample.
3. A coverslip at 45-degree angle was placed.
4. The slide was reviewed under microscope.
RESULT OF THE TEST
•
When the resolution power was at 100x motility of the bacteria could be detected
APPLICATION OF THE TEST
•
The wet mount procedure is performed to observe motile organism.
8
Cell culture
MATERIAL USED
•
Cell Culture Media
Figures: culture media
PRINCIPLE OF THE TEST
Cell culture media provide essential nutrients, growth factors, and hormones required for cell
growth and proliferation. They create an optimal environment for cells to survive and replicate.
PROCEDURE
1. The cell culture media were prepared by combining various components such as amino
acids, vitamins, salts, and sugars in a sterile manner.
2. The media were added to the culture vessel containing the cells of interest.
RESULT OF THE TEST
The cell culture media support the growth and proliferation of cells, allowing them to maintain
their viability and perform desired functions. The result is the successful establishment and
maintenance of a cell culture system.
APPLICATION OF THE TEST
Cell culture media are widely used in research laboratories, pharmaceutical companies, and
biotechnology industries for various applications such as drug discovery, toxicity testing, cellbased assays, and production of recombinant protein.
9
Reference
Dr. abdelraouf A. Elmanama (Ph.D. microbiology) (2009) General Microbiology Laboratory
J.R. Sokatch (1969) Bacterial Physiology and Metabolism
The American Society for Microbiology volume 31 (1993) Journal of Clinical Microbiology
10