Assignment NO 2

Internship
BTI619
Tests of Microbiology Department:
1. Gram’s Staining:
2. Ziehl-Neelson Staining
3. Types of Agar Culture Media for bacteria and culturing (commonly
use in diagnostic laboratory)
1.Gram’s Staining:
Principle
Gram staining is based on the principle of difference between the cell wall of gram Positive and Gram Negative bacteria.
Gram positive bacteria stains with crystal violet and are not decolorized with acetone iodine, while gram negative bacteria
are decolorized with acetone iodine and hence take up the color of the dye carbol fuchsin.
Materials/Apparatus

● Petri plates
● Inoculating wire
● Burner
● Reagents
● Iodine
● Ammonium oxalate crystal violet solution
● Swabs
● Culture Media
Reagents
1. Ammonium oxalate crystal violet solution
2. Iodine solution
3. Iodine acetone
4. Dilute carbol fuchsin

Steps of Procedure
5. Make a smear (from swab, fluid, or culture) and fix it by passing the slide rapidly over the flame.
6. Cover the slide with ammonium oxalate crystal violet and permit it to act for 30 seconds.
7. Pour off and wash freely with iodine solution. Cover with flesh iodine solution and permit to act for 30
seconds.
8. Pour off iodine solution and wash freely with acetone iodine. Cover with acetone iodine and permit to act
for about 30 seconds (Bill stain stops carrying out).
9. Wash thoroughly with water.
10. Counterstain with dilute carbol fuchsin for 30 seconds.
11. Wash with water, blot and dry.
12. See under microscope under oil immersion lens.
Result
● Gram positive bacteria -dark purple
● Yeast Cells -dark purple
● Gram negative bacteria -Pale to red
● Nuclei of pus cells -Red/Pinkish
● Epithelial Cells -Pale red/ Pinkish

And its Interpretation

1. The report should include the subsequent information:
2. The number of bacteria present; (many, moderate, few or scanty).
3. The gram reaction of bacteria (gram positive or gram negative).
4. The morphology of bacteria, (cocci, bacilli, others intracellular or not).
5. The presence and number of pus cells.
6. Presence of yeast cells or epithelial cells.
7. Examples: Gram stained urethral smear shows numerous pus cells and moderate number of gram-negative
diplococci a number of which are intracellular.
8. Quality control: Known gram positive and negative organisms should be stained on an equivalent slide of
test organisms.
 Uses
● This technique is employed to differentiate two large groups of bacteria supported their
different cell membrane constituents.
● The Gram's method procedure distinguishes between gram positive and gram-negative
groups by coloring this red violet.
● A Gram's method test is employed to spot bacteria. it's one among the foremost common
ways to quickly diagnose bacterial infection within the body.
2. Ziehl-Neelson Staining:
Principle
The technique is employed to stain mycobacterium and actinomycetes species. Mycobacterium when stained
with carbol fuchsin resist decolorization by acid while other bacteria get decolorized. Hence these are called
Acid Fast Bacteria or AFB. Mycobacterium leprace is a smaller amount acid fast and is decolorized with 5
salicylic acid .

Materials/Apparatus
1. Petri plates
2. Inoculating wire
3. Burner
4. Reagents
5. Sulphuric acid
6. Methylene blue
7. Ethanol
8. Alcohol
 Tests of Molecular Biology Department:
1. ELISA and its types (Commonly used in diagnostic laboratory)
2. Polymerase Chain Reaction and its types (Commonly used in diagnostic laboratory)
Reagents
● Ziehl-Neelson (strong) Carbol fuchsin.
● Sulphuric acid 20% solution.
● Alcohol 95%.
● Ethanol 95 ml plus water to 100 ml.
● Loeffler’s methylthionine chloride .
Procedure
1. Smear is formed on the slide from swab, fluid, or culture etc. it's dried and glued by rapidly passing over
flame.
2. Cover the slide with filtered carbol fuchsin and warmth until steam rises (Do not overheat). Allow the
preparation to stain for five minutes, heat being applied at intervals to stay the stain hot. Boiling must be
avoided.
3. Wash with water.
4. Cover the slide with 20% vitriol , the red color of the preparation changes to raw sienna .
5. Wash with water after a moment and repeat the above step. Till the film is merely faintly pink.
6. Counterstain with Loffler’s methylthionine chloride for 15-20 seconds.
7. Wash the slide in water.
8. Dry in air and see under the microscope using oil immersion lens.
 Results
● Acid fast bacilli: Red, straight, or slightly curved rods, occurring singly or in small groups.
● Cells: Green or blue.
● Background material: Blue
Interpretation
● If definite bacilli seen, report as “AFB positive” or “Acid Fast Bacilli seen”.
● Quality Control:
● At regular intervals and always when a replacement bath of stain is introduced, two
sputum smears of known high and low AFS positivity should be stained with the routine
smears to see the procedure and interpretation.
 Uses
● This technique is employed to spot acid-fast organisms intrinsically as members of the genus
Mycobacterium .
● Acid-fast organisms are characterized by was-like, nearly impermeable cell walls; they contain mycolic
acid and enormous amounts of fatty acids, waxes, and sophisticated lipids.
● This stain is identified tubercle bacillus , the causative agent of tuberculosis.
● Acid-fast organisms have a lipoid capsule that features a high relative molecular mass and is waxy at
temperature . This makes the organism impenetrate by aqueous-based staining solution.
● These acids resist staining by ordinary methods like a Gram's method .
● It also can be wont to stain a couple of other bacteria, like nocardia.
● Acid-fast bacilli are bright red after staining.
3.Types of Agar Culture Media for bacteria and culturing (commonly use in diagnostic
laboratory)
● Types of Agar Culture Media:
● Blood Agar/Chocolate Agar
● Tellurite agar
● MacConkey Agar
● Deoxycholate Citrate Agar (DCA)
● Thiosulphate citrate salt Agar (TCBS)
● Sabouraud Agar
● DNase Agar

For the identification of bacteria, it's essential to get a culture by growing the organisms on artificial media. If
quite one species or types are present, then repeated sub-cultures are required. within the process of culturing
bacteria there are three steps, which are as follows:
The preparation of an appropriate medium .
The initial removal of other organisms from the medium and its containers by sterilization. Adjustment of pH of
medium.
The culture of the organisms, its isolation and separation from other organisms present within the material.
Types of Culture Media:
● Simple media
● Enriched media
● Selective media
● Differential media
 ELISA and its types

ELISA stands for an enzyme-linked immunosorbent assay and it's a test that detects and measures antibodies in
your blood. This test are often wont to determine if you've got antibodies associated with certain infectious
conditions. In 1974, P. Perlmann and E. Engvall developed the test as a substitute surely radioimmunoassay
tests, and eventually, it replaced the western blot test for HIV confirmation. The ELISA test is flexible and medical
professionals can perform it easily as compared to other more complicated tests; many variations are available
commercially.

Principle
ELISA may be a plate-based assay technique designed for detecting and quantifying peptides, proteins,
antibodies, and hormones. In ELISA, an antigen must be immobilized to a solid surface that's linked to an
enzyme. ELISA tests are generally good and accurate tests. they're considered sensitive and specific (accurate)
and compare favorably with other methods used for the detection of drugs within the body. The ELISA testing
method is more straightforward and easier to perform than older laboratory techniques, which frequently required
radioactive materials.
Materials/Apparatus
1. ELISA readers
2. Pipette
3. Washing system
4. Washers
5. ELISA kits are used

Procedure
● Antibody coating
● Protein capture
● Detecting antibody
● Streptavidin-enzyme conjugate
● Addition of substrate
● Analysis

Results
Test results are reported varies supported the laboratory that conducts the analysis. It also depends on the
condition that you're being tested. Your doctor should discuss your results and what they mean. Sometimes, a
positive result will mean that you simply don't have the condition. False positive and false negative can occur.
Uses
An ELISA test could also be wont to diagnose, HIV, Lyme disease , syphilis, chicken pox etc.
It is used for detecting and quantifying substances like peptides, proteins, antibodies, and hormones.
 According to how it works, ELISA are often divided into four major types:

Direct ELISA (antigen-coated plate; screening antibody):

Attachment of an antigen to a polystyrene plate followed by an enzyme-labeled antibody which will react with the
antigen and a substrate which will be measured.

Indirect ELISA (antigen-coated plate; screening antigen/antibody):

Attachment of an antigen to a polystyrene plate followed by an unlabeled or primary antibody which will react with
both the first antibody and substrate.

Sandwich ELISA (antibody-coated plate; screening antigen):

A capture antibody is attached to the polystyrene plate, then antigen is added that specially attaches or captures
the antigen. A second antibody, also specific for the antigen but not an equivalent because the capture antibody
is added and “sandwiches” the antigen. This second antibody is then followed by an enzyme-labeled antibody
specific for the second antibody which will react with a substrate which will be measured.

Competitive ELISA:
This test is just like the sandwich ELISA but involves the addition of competitive antibodies or proteins when the
second antibody is added. This leads to a decrease within the substrate signal generated. This test gives good,
highly specific results.
Polymerase Chain Reaction and its types
Polymerase chain reaction (PCR) may be a method widely utilized in biology to rapidly make millions to billions of
copies of a selected DNA sample, allowing scientists to require a really small sample of DNA and amplify it to an
outsized enough amount to review intimately . Most PCR methods believe thermal cycling.
The polymerase chain reaction has been elaborated in some ways since its introduction and is now commonly
used for a good sort of applications including genotyping, cloning, mutation detection, sequencing, microarrays,
forensics, and paternity testing.
Principle
PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a
single-stranded DNA template. DNA polymerase adds nucleotide to the 3’end of a custom-designed
oligonucleotide when it's annealed to a extended template DNA. Its principle is predicated on the utilization of
DNA polymerase which is an in vitro replication of specific DNA sequences. This method can generate tens of
billions of copies of a DNA fragment from a DNA extract.
Material/Apparatus
Thermocycler is employed as apparatus.
Spinner and agarose gel electrophoresis unit.
PCR tubes, stands, pipettes, tips.
Template: previously isolated and purified.
Two primers: to flank the target sequence.
Four deoxynucleosides triphosphate (dNTPs): to supply energy and nucleosides for the synthesis of DNA.
Buffer system: containing magnesium.
DNA polymerase.
 Procedure
Thermal Cycling Profile for normal PCR
Denaturation
Annealing
Extension

Denaturation:
The DNA template is heated at 94 degree Centigrade . This breaks the weak hydrogen bonds that hold DNA
strands together during a helix, allowing the strands to separate creating single stranded DNA.

Annealing:
The mixture is cooled from 50-70 degree Centigrade . this enables the primers to bind or anneal.

Extension:
The reaction is then heated to 72 degree Centigrade , the optimal temperature for DNA polymerase to act.

Results:
Run a DNA ladder along side the PCR amplicons in order that we will analyze the results. supported the
migration of DNA fragment within the gel and our in silico PCR or primer 3 results we will assume what size our
PCR amplicons are.
 Uses
1. PCR is employed in analyzing clinical specimens for the presence of infectious agents, including HIV,
hepatitis, malaria, anthrax, etc.
2. PCR can provide information on a patient’s prognosis and predict response or resistance to therapy.
3. PCR is employed within the analysis of mutations that occur in many genetic diseases.
4. PCR is extensively utilized in analyzing clinical specimens for the presence of infectious agents, including
HIV, hepatitis, human papillomavirus (the causative agent of genital warts and cervical cancer), Epstein-
Barr virus (glandular fever), malaria and anthrax.
5. The polymerase chain reaction (PCR) may be a technique widely utilized in biology , microbiology.
genetics, diagnostics clinical laboratories, forensic science, ecology , and lots of other applications.
 Types of PCR:
● Real-Time PCR (quantitative PCR or qPCR)
● Reverse-Transcriptase (RT-PCR)
● Multiplex PCR
● Nested PCR
● High Fidelity PCR
● Fast PCR
● Hot Start PCR
● GC-PCR
● ELISA-PCR
● Assembly PCR
● Long-PCR
● RAPD
● Assembly PCR
● In situ-PCR
● Nested PCR
● Allele specific PCR

The End