PREPARATION OF INOCULUM
Preparatory to the test, inoculate the surface of a suitable volume of solid agar medium from a recently revived stock culture of each of the specified microorganisms. The culture conditions for the inoculum culture are described in Table 2 in which the suitable media are Soybean–Casein Digest or Sabouraud Dextrose Agar Medium .
To harvest the bacterial and C. albicans cultures, use sterile saline TS, washing the surface growth, collecting it in a suitable vessel, and adding sufficient sterile saline TS to obtain a microbial count of about 1 × 108 colony-forming units (cfu) per mL. To harvest the cells of A. niger, use sterile saline TS containing 0.05% of polysorbate 80, and add sufficient sterile saline TS to obtain a count of about 1 × 108 cfu per mL.
Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., Soybean–Casein Digest Broth or Sabouraud Dextrose Broth) and the cells harvested by centrifugation, then washed and resuspended in sterile saline TS to obtain a microbial count of about 1 × 108 cfu per mL. [Note—The estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge microorganisms. Refrigerate the suspension if it is not used within 2 hours. ]
Determine the number of cfu per mL in each suspension, using the conditions of media and microbial recovery incubation times listed in Table 2 to confirm the initial cfu per mL estimate. This value serves to calibrate the size of inoculum used in the test. The bacterial and yeast suspensions are to be used within 24 hours of harvest, but the fungal preparation may be stored under refrigeration for up to 7 days.
The table 2 they reference has a column for incubation temp, inoculum incubation time and microbial recovery time. This is what's throwing me off.
Way I see it, you get a starting population of 10^8 CFU's based on turbidometric measurements (and if anyone knows of a good chart to show this it'd be appreciated) and then you...what? Where do the incubation and recovery times come into play? They say to use the suspensions within 24 hours. How do you use these times to determine the population? I'd just take a sample and do a membrane filtration test on it to get the population, run concurrently with the actual inoculated samples.
Anyone run this before or understand what they are talking about with these "inoculum incubation time" and "microbial recovery times?" I suspect they are making it more complicated than it is.
First time post, hope to scan the archives soon to glean some good info