Blood agar

Mice are utilized for testing antiseptics for appHcation to cuts, wounds, and incisions (339). The test bacteria, type 1 pneumococcus and hemolytic streptococcus, ate appHed to the taHs of anaesthetized mice. The tip of the taH is then dipped into the antiseptic for 2 min, after which one-half inch of the taH is removed and inserted into the peritoneal cavity and the incision is closed. If after 10 days the animals survive, the product is considered satisfactory for use as a skin antiseptic. The blood of dead animals is sampled and streaked on blood agar for confirmation of infection from the test bacteria as the cause of death. Since lack of toxicity is another requirement of a product to be appHed to wounds, this test has been combined with a toxicity test (340). 

Duplicate counts are made using serial dilutions up to 10-6 and drop plates. Solutions are then spotted onto blood agar plates and incubated at 37 °C for 18 hours after which the number of colony forming units is determined. To pass the BA Challenge Test there must be no growth from the aliquots taken at 15 minutes or more from the 1 400 and 1 1600 dilutions. 

When isolates are believed to be S. schenckii, they should be subcultured on an enriched medium such as blood agar to determine whether they are dimorphic that is, whether they grow vegetatively as hyphae at 25°C and as yeast cells at 35°C. The conversion from the mold form to the yeast form is enhanced by incubation of the inoculated medium in a candle jar. 

For bacteriological analyses, the commercially available culture media were used Endo agar, elective saline agar, blood agar, thioglycolic medium and beef extract broth (BEB) with 1% glucose. Chocolate agar was used for the isolation of haemophilic flora. 

Any general purpose, solid bacteriological medium, such as tryp-ticase soy agar or blood agar, can be used. Selective media are... 

Fecal streptococci use direct inoculation of 1 ml into TSB if there is turbidity, transfer into Azide dextrose broth or Azide blood agar and identify using API and/or automated identification system. 

Plate a small amount of stock onto blood agar the day before infection and incubate overnight at 37°C. 

Inoculate a fresh colony from a blood agar plate into THY medium. 

The inoculum concentration should be verified by plating 10-fold serial dilutions onto blood agar and counting after incubating for 24 h at 37°C (see Note 6). 

Apply 100 pi of each dilution to a blood agar plate and gently swirl the plate. 

Plate blood immediately onto blood agar after making 10-fold serial dilutions using sterile PBS. 

Columbia blood agar (Beckton Dickinson, MD, LISA). 

Make 10-fold serial dilutions of peritoneal lavage samples and plate on Columbia blood agar. 

Take the medium from step d of the subculture procedure and centrifuge at 800 g for 15 min. Discard the supernatant. Using a platinum loop, place the sediment on a blood agar plate (Appendix 4) and incubate at 37°C for at least 2 days to check for bacterial growth (Chapter 9). 

Also streak samples onto two blood agar plates (Appendix 4) and two deoxycholate plates. 

Dissolve 40 g blood agar base (Oxoid, Basingstoke) in 1 I of distilled water by standing in a boiling water bath. 

The bacterium is grown on blood agar plates and stored in a mixture of Mueller-Hinton broth containing 20% (v/v) glycerol. In this mixture, bacteria can be stored in small aliquots for several years at -70°C. 

To prepare plate cultures, frozen stocks are placed on dry ice and are stabbed with a hot wire loop. Material adhering to the loop is then plated onto the blood agar plates. Frozen stocks may require several passages to regain growth characteristics. 

To prepare a seeding culture, fill a 25 pL universal with approximately 10 pL of sterile Mueller-Hinton broth and seed these with 1-2 colonies from the blood agar plate. This is then incubated at 37°C overnight with vigorous shaking at approximately 200 rpm. 

Microbiologic culture studies are useful fc>r bacterial identification, especially when an ocular infection foils to respond to treatment. Cultures are often obtained from the eyelids, the conjimctiva, expressed material from the lacrimal sac, and the cornea. Because preserved ophthalmic anesthetics have a bacteriostatic effect, cultures should be obtained if possible before anesthetic instillation. In the case of corneal sampling, it is necessary to provide topical anesthesia for patient comfort. The anesthetic of choice is 0.5% proparacaine because it causes the least bacterial growth inhibition. To enhance the bacterial yield, sterile preservative-free anesthetic may be used. Samples obtained may be inoculated directly onto soUd media plates (e.g., blood agar). Amies without charcoal transport medium (e g., BBL CultureSwab Plus) appears to be an acceptable alternative to direct plating and has the added benefit of convenience. 

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