In this experiment, students should be able to estimate the number of viable aerobic microorganisms present in pharmaceutical products, food, and drinks.
What will students do in this experiment?Students will be given (or they bring their own) a sample of pharmaceutical products, food or drink that will be examined for its number of viable aerobic microorganism. In microbial contamination test, we want to know how much microbial contamination in a products. In limit test, we examined the sample then compare with the limit that state in requirement, then conclude if the products fulfill the requirement or no. Both tests use the same method.
How?If the sample is solid or semisolid, we have to dissolve it first, using certain buffer (see in Pharmacopoeia). If the sample is liquid we continue to next step. The sample or sample solutions then are diluted to certain concentration using NaCl 0.9% solution (with the certain amount for example, we use 1 mL of sample, after that continue with the dilutions). The concentrations used are exponential, 10
-1, 10
-2, 10
-3, 10
-4, ..., ..., ... (start and continue as needed). 1 mL of each concentration is transferred to each Petri dish that already contain Agar (we use NA for bacteria and SDA for fungi). If the materials are enough, do replicate for each series. Then, we incubate all of them (NA in 37
oC and SDA in 25
oC). Do observation for 7 days. Count the colonies formed 7 days of observation.
Pick one Petri Dish that contain colony amount in range 30-300 (
why should 30-300? It
is statistical reason). For example your data is like this: Colonies formed in 10
-1; 10
-2; 10
-3; 10
-4 dilution respectively are 500; 380;
250; 15, then obviously, pick the dilution that produced 250 colonies (10
-3). If the data became like this (in the same order of concentrations), 500; 380;250;70, which data that you pick??? Hmm.. take whole data ranging 30-300 that is 250 and 70. Calculate the cfu for each series. So, you get 25000 cfu/mL from 250 colonies in 10
-2 series and 70000 from the other one. Calculate the mean, you will get 160000 cfu/mL. Don't forget to count the colonies in the replicate series too.. (from the same concentration from the first one)
How to do the calculations?After you get the data, for example 250 colonies (from dilution in 10
-3 1st series) and 239 colonies (from dilution in 10
-3 replicate series). Just simply calculate the average then times with the dilution factor. The average is 244.5, times with 10
3 then you got
244.5 x 103 cfu. Because we use 1 mL of sample then 244.5 x 10
3 cfu is the amount of viable aerobic microorganisms present in 1 mL of sample -->
244.5 x 103 cfu/mL. Simple :D
If you got two data that ranging 30-300, just do calculations for the cfu for each series. So, you get 25000 cfu/mL from 250 colonies in 10
-2 series and 70000 from the other one. Calculate the mean, you will get 160000 cfu/mL. Don't forget to count the colonies in the replicate series too.. (from the same concentration from the first one)
Then what does that number means?That number means, in each mL of products, there's (about) 244.5 x 10
3 microbes that will form colonies. Each product has its own requirement... let say the requirement for our products is:
Viable microbes max. 103-104 aerobic bacteria per mL
Was your sample fulfilled the requirement?
What should I conclude?Read the objective! "to estimate the number of viable aerobic microorganisms present in pharmaceutical products, food, and drinks". Report the number then :D
It's simple, isn't it?
Hope all of you understand and can make a good report of this module and also succeed in the lab exam.. :D
image taken from
University of Cincinnati Clermont College. Open the link please, read the article, hope you will get more understand.
Uhm... where's the negative control?? Not remember..
Figure 1. Protein Electrophoresis Calibration Curve
