BIOLOGY & SCIENCE

article : Oxidase Test

A positive oxidase reaction reflects the ability of a microorganism to oxidize certain aromatic amines, such as tetramethyl-p-phenylene diamine (TPD), producing colored end products. This is due to the activity of cytochrome oxidase (a.k.a., indophenol oxidase) in the presence of atmospheric oxygen.

One use of the test is for the preliminary identification of Neisseria and Moraxella species, which are both oxidase positive gram-negative diplococci.

Procedure: Using a sterile wooden stick, remove 2-3 colonies from each culture to be tested and smear on a piece of filter paper. Add a drop of the spot test (TPD) reagent to each spot. If the organism has oxidase activity, it will turn purple within 30 seconds.
Designed & Maintained by David M. Rollins
Copyright © 2000, D.M. Rollins and S.W. Joseph
Revised: August 2000
URL: http://life.umd.edu

article : Nitrate ReductionTest

Nitrate Broth: some bacteria (e.g., Pseudomonas aeruginosa) have respiratory enzyme systems that can use nitrate as a terminal electron acceptor. The product of the reaction is nitrite. Some of the organisms that reduce nitrate to nitrite will then reduce the nitrite further. In the scheme below, first test for nitrite by a colorimetric test. If this test is negative, it can mean either that nitrite was not reduced, or that it was reduced beyond the nitrite stage. This can be resolved by the addition of zinc dust; if nitrate is still present, the zinc will reduce it chemically to nitrite, which will then by revealed by the colorimetric reaction.

Procedure: To the nitrate broth, after 48 hours of incubation, add 0.2 ml of acid reagent (Solution A), a mixture of acetic acid and sulfanilic acid, and then 0.2 ml of dimethyl-alpha-napthylamine reagent (Solution B). If nitrite is present you will get a red color: this is a positive test for nitrate reduction. If there is no color, pick up some zinc dust on the end of an applicator stick, and add it to the tube. ZINC DUST SUSPENDED IN AIR CAN BE EXPLOSIVE; KEEP AWAY FROM FLAMES! If you get a red color at this stage, what can you conclude? What if no color is obtained?

Designed & Maintained by David M. Rollins
Copyright © 2000, D.M. Rollins and S.W. Joseph
Revised: August 2000
URL: http://life.umd.edu/

article : Know the procedure of Methylene Blue Stain

Methylene Blue is a homologue of Toluidine Blue O. This stain is used to visualize intracellular metachromatic granules.

Procedure:

Smears are fixed with heat and allowed to cool.

Stain with the methylene blue (homologue of toluidine blue O) solution two to seven minutes.

Wash slide and blot dry.

Results: By this method, the intracellular metachromatic granules stain a ruby-red to black color; with the remainder of the cell staining a pale blue color.

Designed & Maintained by David M. Rollins
Copyright © 2000, D.M. Rollins and S.W. Joseph
Revised: August 2000
URL: http://life.umd.edu

article : Minimal Inhibitory Concentration (MIC) (Broth Tube Dilution Method)

The tube dilution test is the standard method for determining levels of resistance to an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time. The lowest concentration (highest dilution) of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC). At this dilution the antibiotic is bacteriostatic.

Additionally, the minimal bactericidal concentration (MBC) can be determined by subculturing the contents of the tubes onto antibiotic-free solid medium and examining for bacterial growth.

Although the tube dilution test is fairly precise, the test is laborious because serial dilutions of the antibiotic must be made and only one isolate can be tested in each series of dilutions.

Procedure:

Number sterile capped test tubes 1 through 9. All of the following steps are carried out using aseptic technique.

Add 2.0 ml of tetracycline solution (100 ug/ml) to the first tube. 3. Add 1.0 ml of sterile broth to all other tubes.

Transfer 1.0 ml from the first tube to the second tube.

Using a separate pipette, mix the contents of this tube and transfer 1.0 ml to the third tube.

Continue dilutions in this manner to tube number 8, being certain to change pipettes between tubes to prevent carryover of antibiotic on the external surface of the pipette.

Remove 1.0 ml from tube 8 and discard it. The ninth tube, which serves as a control, receives no tetracycline.

Suspend to an appropriate turbidity several colonies of the culture to be tested in 5.0 ml of Mueller-Hinton broth to give a slightly turbid suspension.

Dilute this suspension by aseptically pipetting 0.2 ml of the suspension into 40 ml of Mueller-Hinton broth.

Add 1.0 ml of the diluted culture suspension to each of the tubes. The final concentration of tetracycline is now one-half of the original concentration in each tube.

Incubate all tubes at 35^oC overnight.

Examine tubes for visible signs of bacterial growth. The highest dilution without growth is the minimal inhibitory concentration (MIC).
Designed & Maintained by David M. Rollins
Copyright © 2000, D.M. Rollins and S.W. Joseph
Revised: August 2000
URL: http://life.umd.edu

article : Minimal Bactericidal Concentration (MBC)

The Minimal Bactericidal Concentration (MBC) assay is performed as an adjunct to the MIC and is used to determined the concentration of antibiotic that is lethal to the target bacteria in vitro.

Procedure:

From each MIC broth tube without visible growth, aliquot a 100 ml volume of the broth onto Mueller-Hinton agar and spread across the entire surface of the plate.

Record the dilution of the subcultured MIC tube on each plate and incubate at 35^oC until the next lab session.

Following overnight incubation, examine the MBC plates for colony growth or lack of growth for each dilution subcultured. No growth indicates that the antibiotic was bactericidal at that dilution. Growth indicates that the antibiotic was bacteriostatic but not bactericidal at that dilution.
Designed & Maintained by David M. Rollins
Copyright © 2000, D.M. Rollins and S.W. Joseph
Revised: August 2000
URL: http://life.umd.edu