BIOL123

Lab 2 Culturing & Aseptic Technique

PRE-LAB QUESTIONS

1. Proper aseptic technique is crucial to ensuring growth of pure bacterial colonies. What is

one experimental way you can test your practices to confirm that you are using proper

aseptic technique?

a. One way I could test to see if my aseptic technique is working would be when

transferring the bacteria from one plate to another, if there are bacteria on the

plate that I didn’t place there, I will know there was a contamination from either

the air or the method of transfer.

2. In a laboratory setting, what are three ways you can properly sterilize culturing

equipment?

a. Wet heat, such as an autoclave, is one way to sterilize equipment. You can also

use dry heat such as a flame or by baking the equipment first. Another way you

can sterilize equipment is by using chemical solvents like bleach.

3. For each inoculation tool, give one scenario in which use of that tool would be

appropriate.

a. Inoculation loops would be used when transferring microbes from one plate to

another. Sterile cotton swabs are usually used when collecting microbial samples

from surfaces, Inoculation needles are used when collecting microbes from a

defined region. Sterile plate spreaders are used to transfer an even bacterial

lawn.

4. Why don’t microorganisms in cultures exhibit constant exponential growth? What are some steps you could take to extend the lifespan of a microbial culture?

a. Microbes growing on the medium eventually will either run out of nutrients, become poisoned by the buildup of waste, or alter the pH of their environment so much that they can no longer continue to survive. In order to extend the lifespan of a microbial culture, I would replenish growth medium through transfer to a new plate or replace the liquid medium

5. Using a textbook or a reputable online source, describe how lab cultures are maintained in a continual pattern of growth. Focus particularly on those used in biotechnology, such as E. coli, which is used to make human insulin.

a. Growth media helps to maintain a continual pattern of growth. Some common growth media for microorganisms are nutrient broths and agar plates; specialized media are required for some microorganisms

6. Which of these has a constant growth pattern: an open system or a closed system?

a. An open system provides unlimited nutrient allowing microbes to grow constantly. There is a limited nutrient in a closed system, where microbes no longer continue to grow.

7. A human patient represents what kind of system for bacterial infections?

a. A human represents an open system for bacterial infections. We continuously get rid of waste products while consuming fresh nutrients, providing bacteria to grow

8. You’re a physician trying to isolate bacterial colonies from the human gut in attempt to diagnose a gastrointestinal infection. You streak your sample on a growth media containing glucose, amino acids, and salts that contain both sulfur and phosphorous with a pH of 7. You incubate the plates in aerobic conditions at 37 ˚C for three days, at which point you can see clear bacterial colonies forming on the plate. Would you feel confident in stating that you had successfully cultured all the bacteria from your gut sample? Why or why not?

a. There may be bacteria that was not able to grow into a culture because they cannot survive in the pH or the temperature. Also, some colonies take longer than 3 days to be visible.

9. Do some research and try to identify one to three types of microbes cultured on your plates. Using any resources available to you (i.e., a textbook or internet sources such as the CDC website), look up what bacteria are usually found on the areas from which you obtained your samples and the morphological characteristics of their colonies. Describe what morphological traits (i.e., size, shape, arrangement, color, margin, etc.) led to your hypothesized species identification. Be as specific as possible in the microbe type.

a… Shoe from work; Besides E. coli, which is known to cause intestinal and urinary tract infections, the soles of the shoes picked up Klebsiella pneumonia bacteria, a source of wound and bloodstream infections as well as pneumonia, and Serratia ficaria, a rare cause of infections in the respiratory tract and wounds. Yikes! Morphological Traits: Size: Large. Shape: Round (Coccus), morphed (Staphylococcus) (streptococcus), sporadic colonies of (E-Coli Salmonella), Color: Opaque/Tan, Margin: Lobate with raised elevation, and circular/irregular form.

10..For the colonies hypothesized in Question 1, specify which plate and source the microbes came from. Are you surprised to find this type of microbe on this surface? (5 points) Plate #1 and Plate #3 had the most growth with different colonies of bacteria as well as E-coli

a.. I used plate #1 (My Shoe from work) because it mortified me to know how disgusting and dangerous the bottom of my shoes are. I clean and disinfect them all the time, but the sample was taken after one 12hr night shift. Yes, I am very surprised to find these types of microbes on the surface of my shoe. Although this is an expected outcome due to the fact I work in a very busy hospital setting.

11..Looking at your control, did you perform proper aseptic technique or were your plates contaminated? If contamination did occur, list the possible sources and how you can prevent contamination in the future.

a… My plate looked a little bit contaminated. The possible source was that the cotton swab was not sterol even before I opened it form the package, or the prepackaged deionized water that I dipped the cotton swab in wasn’t sterol. I came to these conclusions because in experiment #2 my control samples were clear of contamination.

12.. Was there a large risk for airborne contamination of your experimental plates? Based on the colonies that grew on your plates, do you think any of your experiment plates received airborne contamination?

a… Yes, there is usually a large risk for airborne contamination in these experiments, but this was not the case of my experiment. I had No Growth, No Color, No Amount, No Shape. Clean Clear Plate. Which tells me my experiment was successful.

13.. Were all of your colony transfers successful? Explain what could have been the cause of any unsuccessful transfers.

a.. Yes, all of my colony transfers were successful.

14.. Did you have any growth that was different in appearance from the initial plates? What might account for any differences in growth on the transfer plate/tube?

a.. Yes, all my plates had different growth appearances from the initial plate #1 sample. I believe the differences in growth and shape were due primarily to the different method of transfer used in this experiment. The purpose was to isolate different individual colonies.

15.. Describe why an unsuccessful transfer or growth of the transfer plate/tube differing from the initial plates would be a problem if this experiment were placed in a larger context (i.e., only one step in a longer experiment).

a.. An unsuccessful transfer will show the absence of colonies on the plate and in the stab tube. Overheating can be a factor that can destroy the organism and the morphology of the cell can also be changed in an unsuccessful transfer.

16.. Do some research and describe two or three scenarios in which it would be preferable to use a stab tube vs. a growth plate. (Hint: What do bacteria use to help them move? Can motility be used to help identify many medically important pathogenic bacteria such as the Enterobacteriaceae?)

a. An inoculation needle is used to isolate individual colonies, the needle picks up less microbes, so there is less of a chance of picking up more than one microorganism/bacterium vs. a growth plate sample. The most common methods of transport for bacteria is with the aid of flagella. Many bacteria also use appendages called Pilli to move along a surface. Species are identified in the clinical laboratory by morphological traits and biochemical tests, some of which are supplemented by serologic assessments (e.g., identification of Salmonella and Shigella species). Because of differences in pathogenicity (Escherichia coli) or the necessity to characterize a disease outbreak (Vibrio cholerae, methicillin-resistant Staphylococcus aureus), strains of medical interest are often classified below the species level by serology or identification of toxins. Yes, motility can be used to identify medically important pathogenic bacteria. https://www.ncbi.nlm.nih.gov/books/NBK8406/

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Table 1: Experiment 1 Colony Growth

Plate Number Source Growth (color, amount, shape,

etc.)

1 Cellphone

There are many colonies all the

same yellowish white color. They

are circular with a translucent look

on the outside and opaquer in the

middle

2 Foot

Many colonies, some are yellow,

and some are white. All the

colonies are round and small

3 Table

There are 3 noticeably different

colors. There is white, light yellow,

and a deep yellow or gold. Half of

the culture are very small round

and defined circles; the other half

are very spread out and in almost

a water spill shape

4 Airborne Contamination

There are two different colors,

white, and translucent yellow. The

colonies are very spread out unlike

the first 3 plates. The white

colonies are in a circle, and the

yellow colonies do not have a

distinct shape.

5 Control No growth

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Table 2: Initial Reserved Plate Colony Growth Observations

Plate Sample Sample Appearance (morphology, etc.)

1 White, circular, opaque

2 Gold/Orange, smeared look, translucent

3 Light yellow, circular, opaque

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Table 3: Final Plate and Stab Tube Growth Observations

Sample Form Growth

(yes or no)

Same Appearance

as Initial Plate?

(Yes or No)

Successful

Transfer?

(Yes or No)

1

Plate Yes No Yes

Stab Tube Yes Yes Yes

2

Plate Yes No Yes

Stab Tube No Yes No

3

Plate Yes Yes Yes

Stab Tube Yes Yes Yes

Control

Plate No Yes Yes

Stab Tube No Yes Yes

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