Preparation of Culture

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 Instructions for Good Laboratory Practice and Care of Laboratory Equipment

Correct use and care of the laboratory equipment is considered a fundamental part of good laboratory technique. All students
working in the microbiology laboratory are responsible for maintaining equipment and materials in proper working condition.
Please read over and follow the instructions listed below:

Microscopes
The most critical (and most expensive) piece of equipment in the microbiology laboratory is the microscope. If you expect to
see specimens through the microscope, it must be kept clean and in good condition. You must use the microscope assigned to
your seat. Instructions for the use and care of the microscope can be found in Lab 1 of the lab manual. Report any problems with
your microscope to your instructor. Inoculating loops and inoculating needles

Inoculating loops and needles are used to transfer bacteria into and from culture media. Inoculating loops have a loop at the
end, while inoculating needles end in a point. Inoculating loops are the most common method of transferring bacteria.
Inoculating needles are used when stabbing into a medium during specific inoculation procedures, or when it is necessary to
pick up a small amount of bacteria from one colony on an agar plate without contacting bacteria in other colonies.

Bunsen burners. A Bunsen burner is a source of open flame that is used to sterilize loops and needles, as well as flaming the lips
of test tubes during inoculations. You must always take great care when operating a Bunsen burner! To light the Bunsen burner,
turn the handle of the valve so it is in line with the tubing connecting the Bunsen burner to the gas. Using a striker or a BBQ
lighter, light the Bunsen burner. If the Bunsen burner does not immediately light, turn off the gas and determine the cause of the
problem. NEVER leave the gas on if the Bunsen burner is not lit. DO NOT lean over the Bunsen burner while lighting it.

Petri dishes and test tubes. All materials used for handling or culturing microorganisms are to be disposed of as follows: test
tubes placed in racks in a bin for autoclaving; petri dishes in the other bin for autoclaving and disposal.

Spillage. Any living culture material that is spilled, either on tables or on the floor, is to be treated immediately with disinfectant
and cleaned up with paper towels. Notify the instructor of any spills. The paper towels that you use to clean up the spill should
be placed in the bin with the petri dishes for autoclaving. Prepared slides Prepared slides that are used during the semester
must be returned clean to the trays from which they were taken. Cleanliness of the room Any papers on the floor at the end of
the laboratory period are to be picked up and discarded in the wastebasket. The same is true for your laboratory bench area.

VERY IMPORTANT! DO NOT throw plates, tubes, swabs, slides, pipets, pipet tips, broken glass, etc. into the regular garbage.
These items need to be disposed of properly. Throwing potentially contaminated items into the regular garbage is a safety issue
for students, instructors, lab techs and the cleaning staff. If these items are found in the regular garbage the ENTIRE BAG OF
GARBAGE must be autoclaved before disposal. If you are unsure about where an item should go, always ask your instructor.

General Procedure for inoculating media

1. Sterilize an inoculating loop or needle in the flame of a Bunsen burner. The portion of the loop or needle that will contact the
stock culture or the growth medium must turn bright orange for effective sterilization. For the most rapid sterilization, place the
loop at the top of the inner blue cone of flame—this is where the temperature of the Bunsen burner is the hottest. Remove the
loop from the flame after it is properly heated- keeping the loops in the flame for too long will eventually cause them to crack.

2. If you are picking a colony from a plate, cool the inoculating loop on agar that does not contain any bacterial colonies.

3. Pick a small amount of bacteria (you do not need much). If you are inoculating a tube of broth or an agar slant, remove the
cap of the tube (do not set the cap down on the table) and flame the lip of the tube. Throughout the procedure, hold the tube at
an angle to reduce the probability of particles entering the opening. Insert the loop into the tube and transfer bacteria to the
growth medium. Be careful that only the sterilized part of the loop touches the tube or enters the growth medium.

4. Flame the lip of the test tube before replacing the cap. 5. Sterilize the inoculating loop again.
 Laboratory Activity 1: Testing what have you learned:
Direction: Answer honestly the following questions. You may use the material provided above, but
you must explain your answers in your own words. (Each question is worth five points)

Describe culture media.

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What are the common types of culture media?

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Microbiology Case Studies Using Differential &

Selective Media

Case Study 1

A 28-year-old patient arrives at the emergency room with complaints of redness, pain and streaking along
the site of a surgical incision on the leg. The patient has a fever, and the incision site is leaking purulent
drainage. The patient is scheduled for surgical debridement of the wound, and during the procedure a tissue
sample is collected and sent to the microbiology laboratory for culture. The specimen is plated to standard
microbiology media, including blood agar (trypticase soy agar enriched with 5% sheep blood). After 24 hours
of incubation, small colonies with a large zone of beta hemolysis are growing on the blood agar plate.

Guide Questions:

1. What is/are the media used in the case study 1?

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2. What are your cues in choosing your answer in guide question 1.
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Case Study 2
The microbiology laboratory receives blood cultures on a 50-year-old patient with a variety of underlying,
complex medical conditions. The blood cultures flag positive on the automated blood culture instrument at 18
hours of incubation, and gram-negative rods are seen on the Gram stain. The blood is plated to blood,
MacConkey and chocolate agars and incubated for 24 hours. While the plates are incubating, the clinician
calls the laboratory and says that the medical team is trying to choose appropriate antimicrobials for this
patient, and they would like to know if the organism is Pseudomonas aeruginosa as soon as possible. The next
day, pink colonies are growing on the MacConkey agar.

Guide Questions:

1. What is/are the media used in the case study 2?

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2. What are your cues in choosing your answer in guide question 1?
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REFERENCE
https://asm.org/Articles/2020/September/Why-Differential-Selective-Media-Are-Invaluable-To

https://www.sigmaaldrich.com/PH/en/technical-documents/technical-article/microbiological-testing/microbial-
culture-media-preparation/media-preparation

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/6%3A_Culturing_Microo
rganisms/6.3%3A_Culturing_Bacteria/6.3C%3A_Selective_and_Differential_Media

https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/6%3A_Culturing_Microo
rganisms/6.3%3A_Culturing_Bacteria/6.3A%3A_Culture_Media

https://learn.chm.msu.edu/vibl/content/differential/
https://academicworks.cuny.edu/cgi/viewcontent.cgi?article=1015&context=qb_oers