Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger
Objective: To test an organism's susceptibility to the chemical, optochin. Optochin susceptibility tests the fragility of the bacterial cell membrane. This test is mainly used to differentiate between Streptococcus pneumoniae (sensitive) and other Streptococcus species (resistant)
1. Pick a single pure colony with a sterile swab to inoculate a SBA plate. Streak the entire blood agar plate with the swab. Turn plate 90 degrees and re-streak with the same swab. Blood agar plate must be used for optochin testing since all species of Streptococcus are fastidious organisms and require extra enrichment for growth.
2. With alcohol flamed forceps, aseptically remove an optochin disc and apply to the center of the plate. Gently apply pressure to disc so that it adheres to the surface of the plate but do not press disc down into the medium.
3. Invert plate and incubate for 48 hours at your organism's optimum growth temperature.
-- Sensitive (S): A distinct zone of inhibition (5 to 30 mm) with a clear-cut margin around disc.
-- Resistant (R): Growth not inhibited around disc.
-- Occasionally, a few scattered optochin resistant colonies of S. pneumoniae may be observed in a wide zone of inhibition.
-- Occasionally an alpha-Streptococcus spp. may exhibit a very small zone (1 to 2 mm) of inhibition. S. pneumoniae exhibits a zone of inhibition at least 5 mm or greater in diameter.
Objective: To test an organism's susceptibility to the antibiotic novobiocin.
1. Streak a BHI plate using a sterile cotton swab. Turn the plate 90 degrees and restreak with the same swab
2. Using a pair of alcohol flamed forceps, aseptically place a novobiocin disc in the center of the plate. Apply gentle pressure to disc so it adheres to the surface of the agar but try not to press too much to embed the disc into the agar.
3. Incubate the inverted plate 48 hours at your organism's optimum growth temperature.
Sensitive (S): No growth around disc; clear zone around disc.
Resistant (R): Growth not inhibited; growth around disc.
Objective: To determine the ability of an organism to grow in 7.5% NaCl and ferment mannitol.
Any significant growth indicates the organism is a Staphylococcus species. The phenol red indicator changes to yellow at low (acid) pH, which is a product of fermentation. Therefore, fermentation of mannitol will change the color of agar to yellow. Orange is negative.
Positive: Growth, yellow color (mannitol "+").
Negative: Growth or no growth; red or orange color (mannitol "-").
Objective: Some pathogens are able to produce exoenzymes called hemolysins which lyse red blood cells and thus their action can be demonstrated on a blood agar plate.
1. Using a sterile loop, inoculate a blood plate (SBA) with the pure culture of the organism to be tested using the quadrant method. Also stab the medium in the second quadrant with your loop. (Some hemolysins show their effects better under lower oxygen concentrations.)
2. Incubate for 48 hours at optimum temperature for the organism.
Interpret by noting the reaction around isolated colonies as follows:
Alpha (α) hemolysis: formation of a green or brown zone around the colonies (due to loss of potassium from the red cells).
Beta (β) hemolysis: complete lysis of cells and reduction of released hemoglobin; a clear zone appears around isolated colonies.
Gamma (γ) hemolysis: no hemolytic reaction (no change of the medium surrounding isolated colonies).
-- The reaction should be checked only around isolated colonies. If you do not have isolated colonies on the blood agar, a lighter inoculation should be streaked and the test repeated.
Objective: DNase mediates the hydrolysis of DNA. Methyl Green indicator is stable at pHs above 7.5 but becomes colorless at lower pHs. The hydrolysis of DNA in the agar by bacterial DNase reduces the agar pH.
1. Using a sterile loop, inoculate a DNA+Methyl Green agar plate with the fresh bacterial culture. Use a heavy streak line for each bacterial strain to be tested. Be sure to label the plate bottom properly for each strain.
2. Incubate at 37°C for 48 hrs.
-- The test is positive if clearing develops around the areas of growth. If the color of the agar around the growth is unchanged, the test is negative (i.e., the organism is not able to produce DNase).
Objective: To determine the ability of an organism to ferment (degrade) a specific carbohydrate in a basal medium producing acid or acid with visible gas. The acid would change the color of the medium in a positive test. The following carbohydrate semi-solid media tubes are available at our lab:
1. Using a sterile needle, stab the tube within 1/4 inch of the bottom with medium inoculation.
2. Incubate for at least 48 hrs. Bacteria that are known to be slow growers should be given up to 96 hours.
Positive: Any yellow color (not orange). It does not necessarily have to be the whole tube. A positive result is referred to as ("+") or (A) or (Acid), as fermentation forms acidic products.
Negative: A red, pink or orange color - no yellow at all.
-- Gas production
Positive: Significant bubbling in semisolid medium (one small bubble is generally negative, caused by the stab). Gas may also cause the medium to get separated from tube. Record as (G) for positive gas production.
Negative: No gas bubbles except those produced by stabbing.
Objective: To determine if the organism is capable of breaking down starch into maltose through the activity of the extra-cellular α-amylase enzyme.
1. Use a sterile swab or a sterile loop to pick a few colonies from your pure culture plate. Streak a starch plate in the form of a line across the width of the plate. Several cultures can be tested on a single agar plate, each represented by a line or the plate may be divided into four quadrants (pie plate) for this purpose.
2. Incubate plate at 37 °C for 48 hours.
3. Add 2-3 drops of 10% iodine solution directly onto the edge of colonies. Wait 10-15 minutes and record the results.
-- Positive test ("+"): The medium will turn dark. However, areas surrounding isolated colonies where starch has been hydrolyzed by amylase will appear clear.
-- Negative test ("-"): The medium will be colored dark, right up to the edge of isolated colonies.
Many different tests have been devised over the years for classification of microorganisms into families, genera, species and even subspecies. Some of these tests are quite simple to perform while others are complicated and may require sophisticated equipment. The tests presented here are among the easier ones that are utilized in major clinical laboratories around the world. These tests are ordered alphabetically in this section. Make sure that you read the complete discussion of each test before you start to perform it.
IMPORTANT: Many of the tests mentioned in the following sections are enzymatic reactions. Therefore to get a correct results, one needs to warm up the culture and all test materials to temperatures between 25-40°C for the reactions to proceed. If you have stored your plates, broth culture or test reagents in the refrigerator, you may need to place them at the 37°C incubator for 15-20 minutes before performing the test.
Objective: To determine the ability of an organism to reduce nitrate to nitrite which is then reduced to free nitrogen gas. The nitrogen in nitrate serves as an electron acceptor. The result of the denitrification process is the production of nitrite:
In this case, all the NO3- will be converted to N2 gas which escapes to the atmosphere. We can test for this step by looking for the absence of NO3- through the addition of Zn powder as described below.
1. Inoculate a nitrate agar slant with your pure culture using a sterile loop to transfer a rather heavy inoculum.
2. Incubate at 37°C for at least 48 hours.
3. Add 2-3 drops of Reagent A and 2-3 drops of Reagent B to your tube. Reagent A is 0.8% sulfanilic acid in 30% acetic acid and Reagent B is 0.6% N,N-dimethyl-α-naphthylamine in 30% acetic acid (CAUTION: Reagent B is a potential carcinogen, so work in the hood and avoid inhaling it or allowing for contact with skin; wash hands thoroughly after work).
Reduction of nitrate to nitrite is indicated if a red color develops quickly (within 1-2 minutes). If no color develops, add a very small amount of zinc powder (~20 mg) to the tube containing the reagents. If a pink to dark red color develops after adding the zinc powder within 5 min., the test is negative (nitrate is present and is not reduced by the organism but zinc has reduced it to nitrite). If no color develops, the test is positive (the organism was able to reduce all the nitrate to nitrite and further to N2 which escaped from the tube).
-- If tubes are stored in the refrigerator, they should first be brought back up to the optimum temperature of the growth condition of the organism.
-- When performing the nitrate reduction test using α-naphthylamine, the color produced in a positive reaction may fade quickly. Interpret results immediately, particularly when performing a number of tests.
-- A strong nitrate-reducing organism may exhibit a brown precipitate immediately after the addition of the reagents. This is due to the effect of excess nitrite upon the p-amino group of the azo-dye and may be reduced by using dimethyl-α-naphthylamine.
-- Some organisms are capable of reducing nitrate to nitrite, yet they destroy the nitrite as fast as it is formed, yielding a false negative result. This nitrite destruction is evident in quite a few bacteria, particularly some Salmonella and Pseudomonas spp. and in Brucella suis.
-- Do not use an excess of zinc; if too much Zn is added, the large amount of hydrogen gas produced may reduce the nitrite (formed from unreduced nitrate) to ammonia (NH3) that could result in a false negative reaction or just a fleeting color reaction.