OXIDASE TEST

DESCRIPTION

Some bacteria like Pseudomonas, Pasteurellaceae, Campylobacter, Neisseria, Moraxella, can produce cytochrome C oxidase located in their membrane which can catalyse  the transport of electrons from donor compounds to electron acceptors (oxygen). This respiratory system is present in aerobic bacteria which are capable to use oxygen as the final hydrogen receptor.

In the presence of oxygen the cytochrome C enzyme oxidizes the phenylendiamine reagent, resulting indophenol, a purple-blue compound.

PROCEDURE

There are several ways to perform oxidase test: using filter paper, using a swab or adding the reactive directly on plate. Filter paper method: soak a piece of filter paper in N, N, N', N'-tetra-methyl-p-phenylenediamine dihydrochloride. Pick a colony and dispense on filter paper. Swab method: harvest culture from a solid media then add 1-2 drops of reagent. Direct method: add 1-2 drops of reagent to one colony from an agar plate. For all methods a blue color appears within 1 minute if reaction is positive.

HLA Typing

In 1975, Peter Doherty and Rolf Zinkernagel identified human leukocyte antigens (HLA). HLA are proteins found everywhere in the body except red blood cells. They are especially prevalent in white blood cells. Many types of HLA exist, often varying greatly from person to person. Doherty and Zinkernagel were awarded the 1996 Nobel Prize in Physiology or Medicine for this discovery.

Since many types of HLA exist, scientists can use HLA typing for genetic identification. They are able to compare the types of HLA in different people and determine if the people are related based on similarities between these proteins. HLA have become increasingly important for identifying positive matches between donors and recipients of bone marrow transplants.

The degree of variation among HLA in different people provides for fairly accurate paternity testing, with a power of exclusion from 80 to 90% when combined with blood typing and serological testing. The accuracy of HLA typing increases with the rarity of a tested person's HLA. However, this procedure is normally performed serologically, requiring a relatively large amount of fresh (no more than a few days old) blood. Additionally, the collection process can be considered uncomfortable, especially for children, and cannot be performed on infants under the age of 6 months.

PCR Technique

In 1983, Kary Mullis and members of the human genetics team at Cetus Corporation developed a genetic replication technique called polymerase chain reaction (PCR). After several years of fine-tuning the process, PCR became the most popular DNA replication technique by the 1990s. Kary Mullis was awarded the Nobel Prize in Chemistry for this work in 1993.

The popularity of PCR is based on both the sample size and the processing time it requires. DNA testing utilizing PCR can be performed in a matter of hours with a very small DNA sample.

In PCR, scientists isolate a small amount of DNA (an amount easily obtained from a buccal swab). They then copy regions on the DNA many times to establish large quantities of DNA fragments from the copied regions. PCR can be used to copy any region of DNA.

For PCR use in paternity testing, scientists isolate a small amount of DNA and copy many specific DNA fragments (or loci) that help identify and differentiate people. Fragments from one individual are compared to fragments from other individuals, as done in RFLP, and relationships are determined based on the similarities and differences between the DNA fragments or genetic profiles.

How to Use a Hemacytometer

How to use a hemacytometer

by Heather Buschman

French physiologist Louis-Charles Malassez (1842-1909) studied a lot of things in his life. In dentistry, the residual cells of the epithelial root sheath in the periodontal ligament are now called the epithelial rests of Malassez. A genus of fungi is also named for him, which includes species that can cause dandruff and other skin infections.

Malassez also invented the hemacytometer, the thick glass microscope slide traditionally used to count the number of cells in a given volume of liquid. He made an indentation in a regular microscope slide to form a small space that could hold a few drops of cells in suspension. With etched lines and a known area and depth, the number of cells in that particular volume could be calculated. Since the invention was first used to count blood cells, it became known as the hemacytometer (hem = blood, cyto = cells), sometimes also spelled hemocytometer or haemocytometer.

Over the past few years, more than 4,000 people viewed a Scientist Solutions discussion on how to count cells with a hemacytometer. Even for people who learned cell cultures years ago, remembering the calculation can be tricky. Here’s a quick cheat sheet:

1. Transfer a small sample of cell suspension (~100 μl) to a microfuge tube.

2. Dilute 1:2 with Trypan blue or other cell stain.

3. Carefully transfer approximately 10 μl of cells to one of the semi-reflective panels on a hemacytometer covered with a cover slip. Allow the capillary action of the cover slip to gently draw in just enough liquid to fill the chamber liquid. Do not overload.

4. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of the 4 outer squares in the grid. The square should contain 16 smaller squares.

5. Count the number of live cells in at least 2 of the outer squares. (Dead cells turn dark blue with Trypan staining.)

6. Use the following equation to calculate the number of cells in the original volume:

Some tips for best results:

• Gently pipette the cell suspension up and down to mix well and reduce aggregation before removing a small sample.

• Remove any bubbles in the hemacytometer before counting.

• Count cells touching the top and right borders of each of the 4 squares, but not the cells touching the bottom and left borders.

• If there are less than 100 cells total in all 4 squares, count more squares or start again with a more concentrated cell suspension. If there are too many cells to count accurately, start again by diluting the sample 1:2 or 1:10 before adding Trypan blue.

RFLP Technique

In 1985, Sir Alec Jeffreys developed restriction fragment length polymorphism (RFLP), which quickly became the standard technique for DNA testing throughout the 1980s. RFLP provided the world with the first form of genetic testing based on DNA, the body's genetic material.

Each person inherits a unique combination of DNA from both biological parents, and this DNA serves as the code for all of that person's biological characteristics. By comparing the unique genetic code of one person to that of an alleged relative (for example, comparing a child to an alleged father), one can see whether or not the two people are biologically related.

Using RFLP, scientists cut specific portions of DNA into fragments for comparison. These fragments have different lengths, depending on the location of certain markers for cutting the DNA.

In a family relationship test, one person's DNA fragments are compared with those of an alleged relative. If the fragments match each other, they are considered to be from biologically related people.

In the case of a paternity test, a child's DNA fragments would be compared to the fragments of the mother and alleged father. Since the child received half of his or her DNA from the mother and the other half from the father, his or her DNA fragments should match those of both parents. Half should match the mother's, and half should match the father's. If the alleged father is not the child's biological father, the child's DNA will not match the father's.

In some RFLP cases, it will appear that the child's DNA does not match either parent's; this situation requires extra analysis. Occasionally, mutations in the DNA occur, causing a mismatch of fragments. When this happens, statistical analysis is needed to determine the likelihood of a mutation and the biological relationship between the family members.

Despite the periodic complication of genetic mutations in this procedure, RFLP is highly accurate, with a power of exclusion of 99.99% and higher.

RFLP is a highly accurate test. However, because it requires a large amount of blood and a long processing time, it is not used as frequently today as it once was.

INDOLE PRODUCTION

DESCRIPTION

Indole is an aromatic heterocyclic organic compound that can be produced by some bacteria as a degradation product of the amino acid tryptophan. Tryptophanase is the enzyme involved tryptophan degradation resulting indole, pyruvate, and ammonia. Indole test is most used for coliforms identification.

MATERIALS
Simplest medium for indole testing is Buffered peptone water (Proteose peptone 10g, NaCl 5g, Disodic phosphate 3.5g, Monopotasic phosphate 1.5g, H2O ad 1000 ml; pH 7.2), but most used for Enterobacteriaceae identification is MIU medium (Motility Indole Urea).

PROCEDURE
Inoculate a Buffered peptone water medium or a MIU tube and incubate  at 37°C. After 24 hours of incubation add few drops of Ehrlich-Kovacs reagent. This will combine with the indole forming a red layer on the surface of the medium, if the reaction is positive. Reaction is negative if no red color appear.

NOTES
Indole should not be tested after 4-5 days of incubation because bacteria may further degrade indole and a false negative result may occur.

REFERENCES
1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.

COAGULASE TEST

DESCRIPTION

Coagulase is an enzyme produced by some bacteria that reacts with prothrombin in the blood, forming a complex named staphylothrombin, which causes blood to clot by converting fibrinogen to fibrin. This mechanism protect bacteria from phagocytosis. Frequently coagulase test is used to distinguish between different types of Staphylococcus isolates.

PROCEDURE

Harvest blood from  a rabbit using EDTA to prevent coagulation. Separate plasma from blood cells deposit. Inoculate rabbit plasma with bacteria culture and incubate the tube in a 37 °C incubataor for 3 hours. Examine hourly the clot formation. If negative then continue incubation up to 24 hours.

Result is positive if the serum coagulates resulting a clot (or a solid block when the reaction is very intense). Handle the tube with care. The clot may be fragile and may be disrupted before reading the reaction.

Result is negative if  the plasma remains liquid.

REFERENCES:

NOTES

Utilization of 2% Na citrate for blood harvesting is no longer recommended. Some bacteria can utilize citrate from plasma and false positive reactions may appear.

1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.

2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown Publishers.

Types of microscopes

There are several types of microscopes available on the market, selection of the proper type is not a simple assignmen as you would need to determine what exactly it would be used for. Below you can see all the types of modern microscopes for any scientific and hobby task.


A compound microscope is an optical device made for magnifying objects, consists of a number of lenses forming the image by the lens or a combination of lenses positioned near the object, projecting it to the ocular lens / lenses or eyepieces. The compound microscope is the most used type of a microscope.


An optical microscope, also called "light microscope", is a type of a compound microscope that uses a combination of lenses magnifying the images of small objects. Optical microscopes are the oldest type and simplest to use and manufacture.


A digital microscope has a digital CCD camera attached to it and connected to a LCD or a computer monitor. A digital microscope usually has no eyepieces to view the objects directly. The trinocular type of digital microscopes have the possibility of mounting the camera, that would be an USB microscope.


A fluorescence microscope or "epifluorescent microscope" is a special type of a light microscope, instead of light reflection and absorption used fluorescence and phosphorescencea to view the samples and their properties.


An electron microscope is one of the most advanced and important types of microscopes with the highest magnifying capacity. In electron microscopes electrons are used to illuminate the tiniest particles. Electron microscope is a much more powerful tool in comparison to commonly used light microscopes.


A stereo microscope, also referred to as "dissecting microscope", uses two objectives and two eyepieces which makes it possible to view a specimen under angles to the human eyes forming a stereo 3D optical vision.

Bacteria - Identifying And Classifying Bacteria

The most fundamental technique for classifying bacteria is the gram stain, developed in 1884 by Danish scientist Christian Gram. It is called a differential stain because it differentiates among bacteria and can be used to distinguish among them, based on differences in their cell wall.

In this procedure, bacteria are first stained with crystal violet, then treated with a mordant—a solution that fixes the stain inside the cell (e.g., iodine-KI mixture). The bacteria are then washed with a decolorizing agent, such as alcohol, and counterstained with safranin, a light red dye.

The walls of gram positive bacteria (for example, Staphylococcus aureus) have more peptidoglycans (the large molecular network of repeating disaccharides attached to chains of four or five amino acids) than do gram-negative bacteria. Thus, gram-positive bacteria retain the original violet dye and cannot be counterstained.

Gram negative bacteria (e.g., Escherichia coli) have thinner walls, containing an outer layer of lipopolysaccharide, which is disrupted by the alcohol wash. This permits the original dye to escape, allowing the cell to take up the second dye, or counterstain. Thus, gram-positive bacteria stain violet, and gram-negative bacteria stain pink.

The gram stain works best on young, growing populations of bacteria, and can be inconsistent in older populations maintained in the laboratory.

Microbiologists have accumulated and organized the known characteristics of different bacteria in a reference book called Bergey's Manual of Systematic Bacteriology (the first edition of which was written primarily by David Hendricks Bergey of the University of Pennsylvania in 1923).

The identification schemes of Bergey's Manual are based on morphology (e.g., coccus, bacillus), staining (gram-positive or negative), cell wall composition (e.g., presence or absence of peptidoglycan), oxygen requirements (e.g., aerobic, facultatively anaerobic) and biochemical tests (e.g., which sugars are aerobically metabolized or fermented).

In addition to the gram stain, other stains include the acid-fast stain, used to distinguish Mycobacterium species (for example, Mycobacterium tuberculosis, the cause of tuberculosis); endospore stain, used to detect the presence of endospores; negative stain, used to demonstrate the presence of capsules; and flagella stain, used to demonstrate the presence of flagella.