BIOL123

“Lab 2 Culturing & Aseptic Technique BIO250L”

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“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? While moving bacteria from a given plate to the other, one way to whether if the aseptic method is working properly is to check for bacteria on the plate that was done not put there. If the bacteria is in the plate, I will absolutely identify there was infection from the air or the way of moving.

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

a. The ways to properly sterilize equipement is application of wet heat, like an autoclave.

b. Dry heat, such as a flame, can also be utilized, or the apparatus can be baked beforehand.

c. Chemical solvents like bleachcan also be utilized to sterilize culturing equipment.

3. For each inoculation tool, give one scenario in which use of that tool would be appropriate. While moving microbes from one bowl to another, immunization loops may used. Innoculation needles are used to gather germs from a designated zone and sterile cotton swabs are used to collects microbial from surfaces. To transmit an even bacterial lawn, sterile plate apreaders are used.

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? Microorganisms budding on the agent will eventually exhaust nutrients, get polluted by waste buildup, or modify the PH of its environments to the extent where they cannot thrive. To extend life of a bacterial culture, I would replace the growth media by moving it to a new plate or replace the liquid agent.

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. Growth culture contributes in the maintenance of a steady pattern of growth. Nutrient potages and agar bowl are common microbe growth medium; nevertheless, certain germs require specific media.

6. Which of these has a constant growth pattern: an open system or a closed system? An open system offers an unlimited source of nutrients, letting bacteria to flourish indeterminately. In a locked system, where bacteria will never grow, there is a restricted source of nutrients.

7. A human patient represents what kind of system for bacterial infections? A person is an open system for bactierial diseases. We are contimually removing surplus while eating new nutrients, which allows 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? It’s possible that certain bacteria couldn’t develop into aculture because they couldn’t withstans the PH or temperature,. In addition some groups take longer than three days to appear.

“EXPERIMENT 1: Agar Plate Preparation and Bacterial Inoculation

Data Tables

Table 1: Experiment 1 Colony Growth

Plate Number	Source	Growth (Color, Amount, Shape, etc.)
1	CONTROL	NONE GROWTH FOUND
2	AIRBORNE-CONTAMINATION	SMALL WHITE ROUND GROWTH
3	BATHROOM DOOR KNOB	MANY SMALL AND WHITE SPOTS IN THE BOWL
4	TRASH CONTAINER COVER	BIG SPIDERWEB AT THE CENTER
5	CAR GEARSHIFT	LARGE WHITE GROWTH. IT APPEARS TO BE CONSTANT OF SNOT.

Post-Lab Questions

1. 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. For example, work shoes, in addition to E coli, which can cause intestinal and urinary tract infections, Klebsiella pneumonia bacteria, which can cause wound and bloodstream infections as well as pneumonia, and Serratia ficaria a rare cause of infections in the respiratory tract and wounds, were found on the soles of the shoes.

2. 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? Plate # 1 ( work shoe) because it mortified me to know how disgusting and dangerous the bottom of my shoes are. The sample was taken from my work shoes, I am so surprised to find these types of microbes on the bottom surface of my shoe. Althought this is expected outcome due to the fact I work in a very busy office area.

3. 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. My dish appeared to be polluted; The cotton swab may have been sterol free even before I opened the container, or the supplied deionized water in which I dipped the cotton swab could have been sterol free. These conclusions were reached because my control samples in experiment # 2 were free of contamination.

4. 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? Yes, there is normally a significant danger of airborne contamination in these tests, however that was not the case in my situation. I didn’t have any growth, color, quantity or shape. Plate should be clean and clear. Therefore my experiment was a success.

Insert a photo of your plates after incubation. Include your name and access code handwritten in the background of your photo.

“EXPERIMENT 2: Bacterial Transfer to a Stab Tube and an Agar Plate

Data Tables

Table 2: Initial Reserved Plate Colony Growth Observations

Plate Sample	Appearance (morphology, etc.)
1	Light orange, round small
2	A spiderweb like
3	Light yellowish, round milky shape, large

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
1	Stab Tube
2	Plate
2	Stab Tube
3	Plate
3	Stab Tube
Control	Plate
Control	Stab Tube

Post-Lab Questions

1. Were all of your colony transfers successful? Explain what could have been the cause of any unsuccessful transfers. Yes, All the colony transfers became successful

2. 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? Yes, all of my plates development patterns differed from the first plate # 1 sample. The changes in development and morphology, I assume, were caused mostly by the various technique of transfer utilized in this experiment. The goal was isolate several individual colonies.

3. 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). The absence of colonies on the plate and in the stab tube indicates a failed transfer. In a failed transfer, overheating can be a component that kills the organism, and the shape of the cell can also be altered.

4. 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?) 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. Therefore 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 theclinical laboratory by morphological traits and biochemical tests, some of which are supplemented by serologic assessments( e.r., 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.

Insert a photo of your plates and stab tubes after incubation. Include your name and access code handwritten in the background of your photo.