Aseptic Techniques

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Objectives

1.- Be aware of where bacteria can be found, and what are the environmental factors that influence the growth of bacteria.

2.- Understand the process and importance of labeling and incubating agar plates properly.

3.- Learn the aseptic techniques practices necessary to prevent cross-contamination

4.- Identify growth of bacteria on the surface of an agar plate, a nutrient broth, and an agar slant.

5.- Use colony number estimates to reach conclusions about amount of bacterial growth.

6.- Determine the effects of handwashing on bacterial abundance. Determine the effects of bench disinfection on bacterial abundance.

7.- Define: pure culture, contamination, aseptic techniques.

8.- Know the characteristics of broth medium, bacterial slants, and agar plates.

9.- Identify the steps of transferring from broth to broth, slant to slant, and broth to plate.

1.- Introduction (4.0 pts.)

1.- ( 1 pt.) Explain the following concepts

What does the phrase ‘bacteria are ubiquitous’ means?

What is media?

2.- ( 1pt.) List two locations in a laboratory where bacteria can be found.

List two places, in a laboratory, where bacteria should never be found.

3.- (1pt.) The first step in working with any type of medium, is to label it properly prior to use.

Why are agar plates always labeled at the bottom?

Agar plates are always incubated upside down. Explain why.

4.- (1 pt.) Incubation temperatures are always given in degree Celsius. The standard incubation temperature of bacteria isolated from human specimens is:

Incubating bacteria, isolated from human specimens, at lower temperatures will slow down their growth. Give the approximate temperature (in degree Celsius) of the following incubators:

Room temperature incubator:

Refrigerator temperature incubator:

2.-

Ubiquity Exercise

(5.0 pts.)

In this exercise bacterial presence was investigated in four different environments: banana, soda can, keyboard, and cash.

1.- (0.5 pts.) What implement was used to collect the bacterial samples from the 4 different objects?

Name the type of medium used in this exercise to grow the bacteria.

2.- (1 pt.) Mention two steps that prevented cross-contamination during this exercise.

3 .- (1 pt.) During this exercise, what does it mean to ‘streak a plate”?

What is a ‘colony’? Are all colonies alike?

Where was the cotton tip discarded after each use?

Do you think that the medium used to dip the cotton tip was sterile? Explain your answer.

4.- (1 pt.) ***Take a picture of the results obtained and upload the picture onto your lab report.

4.- (continuation) Complete the table below with the results of the exercise. Note in the chart under ‘Colony variety’ whether the colonies growing on each plate all look the same, or if there are variations in size, consistency, and color.

NUMBER OF COLONIES PER PLATE

PLATE LABEL
Amount of bacteria
Colony variety seen/not seen

5.- (0.5 pts.) Using the results obtained, explain how handwashing affects bacterial quantity and diversity.

6.- (0.5pts.) Using the results obtained, determine which of the 4 objects (soda can, banana, keyboard, and cash) tested had the most amount and variety of bacterial growth. Were the results expected?

7.- (0.5pts.) Using the results obtained, explain the effects of ethanol disinfection on the bacterial load and diversity of the lab bench.

Is wiping the lab bench with ethanol a method of disinfection, or sterilization? Explain your answer.

3.-

Aseptic Transfer

(3 pts.)

1.- ( 1 pt.) The use of aseptic techniques is important in obtaining pure cultures.

What are aseptic techniques?

What are pure cultures (be specific)?

2.- ( 1 pt.) What can happen to a culture if aseptic techniques are not followed?

What inoculating tools can be used to transfer microorganisms?

3.- (1 pt.) When is the inoculating loop sterilized during a bacterial transfer?

When is the mouth of the culture tube passed through the flame during a bacterial transfer?

4.-

Transfer of E. coli from a broth culture to a sterile broth tube

(3 pts.)

1.- (1 pts.) Put the steps of bacteria transfer from broth to broth in numerical order. * Note, some steps may be used more than once.

——— Remove cap of sterile broth

——– Remove a loopful of bacteria from the stock culture tube

——– Heat the mouth of the stock culture tube.

——– Replace cap of newly inoculated broth

——– Replace the cap of stock culture tube

——– Heat and cool inoculating loop

——— Insert loop with bacteria into the sterile broth

——— Remove cap of stock culture tube

——— Heat inoculating loop to sterilize before placing back into receptacle

——– Heat mouth of newly inoculated broth

——— Heat mouth of sterile broth

——— Label sterile tube with name of organism

2.- (1 pt.) What is the purpose of the negative control tube?

Why is the loop not sterilized immediately before picking up the bacterial sample?

3.- (1pt.) Describe the results obtained.

5.-

Slant culture to sterile agar slant

(2 pts.)

1.- (1pt.) Read the steps outlined below describing a slant-to-slant transfer.

Step 1: Remove cap of bacterial culture, and heat mouth of tube

Step 2: Heat and cool the inoculating loop and remove a small inoculum of bacteria from cultured tube.

Step 3: Heat the mouth of the bacterial culture tube, and replace the cap

Step 4: Heat and cool the inoculating loop and remove the cap from the sterile slant

Step 5: Streak the sterile slant surface with the loop.

Step 5: Heat sterilize the inoculating loop and replace into receptacle

Step 6: Heat the mouth of the newly inoculated slant, and replace cap.

Step 7: Incubate newly inoculated slant, and negative control tube.

Determine what is wrong with the steps and explain what would happen if the procedure is followed as written.

2.- (1 pt.) Describe the results obtained.

6.-

Broth Culture to Sterile Agar Plate and Broth Culture to Sterile Agar Slant

(3 pts.)

1.- (1pt.) After washing hands and labeling materials, outline the steps followed for a successful broth to agar plate transfer of bacteria.

2.- (1 pt.) Describe the results obtained for the nutrient agar plate (NA plate) and the nutrient agar slant (NA slant) transfers.

3.- (1 pt.) Explain two mistakes during a transfer that may result in contamination.

7.- REVIEW (5 xtra pts.)

1.- (1 pts.) List the type of media used in these exercises.

2.- ( 1 pt.) Which of the media used during these exercises provide a surface for colony growth?

Which type of medium provides the most surface area for observing the growth characteristics of bacterial colonies?

3.- (1 pt.) List two advantages of using agar instead of gelatin as a solidifying agent.

4.- (0.5 pts.) How many hours incubation is generally needed to obtain observable bacterial growth?

5.- (1.5 pts.) Name the organism used during these exercises. Identify the genus and species of the organism. Look up its gram reaction, and shape

 

Last Updated on February 7, 2021