ELN Lab 9 5-28 Enumeration of Microbes (Cameron).docx

7 Pages
Unlock Document

University of Florida
Microbiology and Cell Science
MCB 3020L

Chloe Evetts (8990-4674) Cameron Jacobs Enumeration of Microbes – Using Petri Films Purpose The purpose of this lab is to learn the principles of determining how many bacteria exist in a sample. This is a very important part of microbiology because in order to develop new drugs, it is crucial to understand the effect of the drug on the survival of the organism being tested. Knowing how many bacteria exist in a sample is used in many facets, and in this lab, we will perform an expiment using Petri Films to accomplish this. Hypothesis By the end of the lab we will be able to successfully be able to determine how many bacteria exist in a sample. The amount of bacteria will be dependent on different parameters, such as oxygen, temperature, pH, salinity, radiation, light, nutrients available, pressure and others. Some bacteria will do better in certain conditions than others. However, bacteria are highly adaptable, so what may harm them in one experiment may not harm them as much if the experiment is repeated. By using Petri Films, we should be able to correctly identify how many bacteria were present in the original sample by counting the diluted sample and performing the correct calculations Procedure Experiment 2: Plate Count Method Materials per group • Your own saliva • phosphate buffered saline (PBS) or water – 10 ml • P1000 and P200 pipettes and pipette tips • Eppendorf tubes (9) • 3M™ Petrifilm™ Aerobic Count Plates (5) and agar plates (5) • Vortex • Sharpie • “Hockey stick” and beakers with ethanol Note: Petrifilm and agar plates can be used in this experiment interchangeably Saliva is a biological sample and can be a potential biohazard Procedure – work in pairs (one group will use petrifilm, the other group will use agar plates) • Initial, date the top of 5 petrifilm™ plates with your initials and the following dilutions: 10 , 10 , 10 , 10 , and 10 -8 • Label 5 agar plates the same way as the petrifilm -1 -2 -3 -4 • Label 8 Eppendorf tubes with the following dilutions: 10 , 10 , 10 , 10 , and 10 , 10 , 10 , and 10 -8 • Design your 1:10 dilutions, consider what final volume you need to make for the plating either on petrifilm (1 ml) or on agar plates (0.1 ml) - - - - - - -7 - Dilution 1 = 10 2 = 10 3 = 10 4 = 10 5 = 10 6 = 10 7 =10 8 = 10 Final Volume 1 part (sample ) 9 parts (diluent ) • Collect some of your spit (saliva) in a new Eppendorf tube – this is your undiluted sample. • Make 1:10 dilutions dilution series of your saliva. • Using the same pipette, repeat this process until you complete the dilution series. -8 • After the last tube has been diluted, start with the 10 dilution tube, vortex, remove the cap and aseptically transfer 1mL onto the petrifilm™ plate labeled 10 . Spread out the liquid after you close the petrifilm. • From the same tube pipet 0.1 ml onto the corresponding agar plate and spread the liquid around the whole plate with a hockey stick. • Then inoculate the remaining petrifilm™ and agar plates in the same way, moving from highest dilution to lower dilution, transferring 1mL and 0.1 ml respectively from the 10 10 -7 -6 -5 -4 10 10 dilution tubes. You can use the same tip for the procedure. • Stack the petrifilm™ and agar plates in groups of 5 and incubate for 24 to 48 hours at 35-37C Next lab: • A red indicator dye in the plate colors all colonies red. Look for red colonies on your petrifilm. Regular colonies on your agar plates. • Count the colonies and multiply by the dilution factor to determine the original number of bacterial in your sample. • Count only the plate with numbers between 30-300. If less colonies than 30 are encountered you plate is considered TFTC (too few too count), if there are >300 colonies, it’s considered TNTC (too numerous to count) • Record the results calculation and your conclusion in your ELN. Take a picture of the petrifilm and add to your ELN. Dilution factor Dilut
More Less

Related notes for MCB 3020L

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.