Aseptic Technique

STUDENT HANDOUT:

• Aseptic Technique Student Handout

LABORATORY REPORT FORM:

• Aseptic Technique Laboratory Report Form

INTRODUCTION:

When microorganisms are found in nature, they usually exist with other microorganisms and not as a single species. For example, a pinch of soil or a drop of lake water will probably contain many types of bacteria, viruses, yeast, molds, algae, and protozoa.

When working with microorganisms in the laboratory, we need to work with one single species of organism. There are many ways to isolate or separate the various types of microorganisms to obtain what is called a pure culture, or a culture of only one genus and species of organism. These methods are called "pure culture techniques." When you try to isolate an organism, and  also when the organism is growing as a pure culture, it  is important  that  other organisms-from  the air, your hands, the table-do not contaminate this culture.  To make sure of this we use aseptic or sterile techniques to handle micro­ organisms. Aseptic technique, or sterile technique, is the procedure designed to keep unwanted microorganisms from contaminating sterile materials or pure cultures of microorganisms. To facilitate this technique, all materials and media must be sterilized prior to use.  Culture media and glassware are sterilized in an autoclave at 121°C, 15 pounds pressure, for about 15 minutes.

When bacteria or other microorganisms are grown in the laboratory, we need to provide nutrients (carbohydrates, proteins, vitamins, fats) to allow them to grow and reproduce. These nutrients are found in laboratory culture media and can be thought of as food for these organisms. The culture medium used can be a liquid called broth or a solid surface. The solid medium is simply broth to which agar, a hardening agent, has been added. Liquid agar poured into a test tube and allowed to harden at an angle is called a slant. Liquid agar poured into a petri dish provides a large surface on which organisms can grow.

Organisms also must be given oxygen (if they need it), the proper humidity, and a temperature similar to their natural habitat.

When bacteria grow on a solid surface, the number of cells increases until a visible mass of cells, called a colony, appears. Bacteria grow in different ways, and this difference in colony morphology, as it is called, helps us identify the type of bacteria. For example, some growth may be smooth or shiny, some even or jagged, and some may have different colors and textures. A variety of terms describe how organisms grow in a liquid (Figure 1) and on solid surfaces (Figure 2). Use these terms to describe the results of the experiment that follows. 

Figure 1. Various growth in liquid cultures.

Figure 2.  Colonies are described according to color, form, elevation and margin.  Image from Wikimedia Commons.

OBJECTIVES:

1. Inoculate tubes of liquid media and slants.

2. Inoculate plated media to obtain isolated colonies of bacteria.

3. Recognize bacterial growth on plates, in broth, and on slants.

4. Demonstrate knowledge of, and consistently practice, aseptic technique.

PRECAUTIONS:

1. Always wear a disposable apron.

2. Wash your hands with an antimicrobial soap such as liquid Dial®.

3. Wipe tabletops thoroughly with Lysol®.

4. Be extremely careful when using the Bunsen burner.

5. Once the inoculating loop is sterilized, do not touch it to the table, your hands, or any other surface.

6. Do not leave lids open longer than necessary. (Do NOT put lids on the lab table.)

7. Never pick up test tubes by the lid.

8. Discard all materials in biohazard containers for proper disposal.

9. Autoclave ALL materials as soon as they are no longer needed.

10. Never discard cultures in the trash can.

MATERIALS (Work Individually):

1. Plate Cultures of:

   • Escherichia coli [E. coli]

   • Micrococcus luteus [M. luteus]

   • Rhodospirillun rubrum [R. rubrum]

2. (A package of bakers’ yeast can be substituted if bacterial cultures are unavailable.)

3. 1 sterile tube of Nutrient broth 

4. 1 sterile slant of Nutrient agar

5. 1 sterile plate of Nutrient agar 

6. Bunsen burner [if using metal inoculating loop] and striker or sterile, plastic disposable loops

7. Glass marker

8. Test tube rack

9. Incubators:  25°C and 37°C already at these temperatures

10. Biohazard bag

11. Masking tape

You can watch a video of how to light a Bunsen Burner using the link below. 

NOTE: 

For each of the media inoculated, your teacher will incubate one uninoculated tube or plate at the proper temperature as a control.

PROCEDURE #1:  PLATE CULTURE --> BROTH

You can watch a video of how to inoculate broth tubes using the link below.

1. Obtain a tube of sterile broth and mark on it the name of the organism you chose for your cultures, the date, and your initials.

2. Gripping the handle of the loop like a pencil, hold the loop in the Bunsen burner flame to sterilize the wire portion. Heat for at least 5 seconds after it glows red. (This step is unnecessary if a presterilized, disposable plastic loop is used.)

3.  Allow the loop to cool for at least 5 seconds. Do NOT blow on it.

4.  Open the plate culture like a clamshell and remove a small amount of growth using the cooled sterile loop. Close the lid.

5.  Remove the tube cap, holding it in the same hand as the loop. Do NOT place the cap on the table at any point.

6.  Briefly flame the mouth of the tube.

7.  Dip the loop containing the culture into the broth and mix it to loosen the bacterial cells.

8.  Flame the mouth of the tube, replace the cap, and place the tube back into the test tube rack.

9.  Reflame the loop as in step 2 (unless disposable loop is used-then put into a biohazard bag and autoclave or incinerate completely).

10.  Incubate the M. luteus and R. rubrum at 25oC and the E. coli at 37oC.

PROCEDURE #2:  PLATE CULTURE --> SLANT

You can watch a video of how to inoculate agar slants from broth using the link below.

PROCEDURE #3:  STREAK PLATE

You can watch a video of how to prepare streak plates using the link below.

There are different methods for streaking agar plates to achieve isolated colonies.  It depends on what you feel most comfortable using!  Microbiologists typically use this method to produce pure cultures from a mixed sample. The goal of this technique is to spread individual microorganisms so far apart on the surface of a solid medium that they do not touch one another.

Technique #1:

1. Turn the plate upside down and mark the bottom of the dish (the half that contains the agar) as shown in the diagram below. 

2.  Also label the bottom of the plate with your name, the strain and the date.  Never label the cover of an agar plate since covers can be accidentally swapped!

3.  Gripping the handle of the loop like a pencil, hold the loop in the Bunsen burner flame to sterilize the wire portion. Heat for at least 5 seconds after it glows red. (This step is unnecessary if a presterilized, disposable plastic loop is used.)

4.  Allow the loop to cool for at least 5 seconds. Do NOT blow on it.

5.  Open the plate culture like a clamshell and remove a small amount of growth using the cooled sterile loop. Close the lid.

6.  Open like a clamshell the sterile plate to be inoculated.  Streak the loop back and forth in a zigzag pattern over area “A.”  Do NOT lift the loop from the agar and do NOT dig into the agar surface.  Close the lid when you are done.  

7.  Reflame the loop (unless disposable­ loops are used - then put into a biohazard bag and autoclave or incinerate completely).

8.  While the loop is cooling, rotate the plate.

9.  Once the loop has cooled, streak from area “A” into area “B.” Streak the loop back and forth in a zigzag pattern over area “B.”  Do NOT lift the loop from the agar and do NOT dig into the agar surface.  Close the lid when you are done. 

10.  Flame your inoculating loop and allow it to cool (unless disposable­ loops are used-then put into a biohazard bag and autoclave or incinerate completely). 

11.  Once the loop has cooled, repeat the procedure by streaking from area “B” into area “C.” 

12.  Flame your inoculating loop and allow it to cool (unless disposable­ loops are used-then put into a biohazard bag and autoclave or incinerate completely).

13.  Once the loop has cooled, repeat the procedure by streaking from area “C” into area “D.”

14.  Close the lid and tape it to the base of the plate, using two small pieces of tape. (To allow air to circulate, do not completely seal the opening between lid and base.)

15.  Incubate the plate (agar side up) at the proper temperature for 48 hours.

Technique #2:

The same procedure can be done by dividing up the plate into 4 quadrants as shown below (Figure 3). The same procedure as described above would be followed, streaking from quadrant 1 → quadrant 4.

Figure 3.  Streaking onto an agar plate using the 4-quandrant streak plate.

Technique #3:

The same procedure can be done by dividing up the plate into 3 sections (rather than 4).  This is known as the T-streak (Figure 4).  The same procedure as described would be followed, streaking from area 1 → area 3. 

Figure 4.  Streaking onto an agar plate using the T- streak plate. 

Image by Brian M. Forster (ASM MicrobeLibrary)

OBSERVATIONS/CONCLUSIONS:

NOTE: 

Use the uninoculated control tubes and plates for comparison with the inoculated materials.

1. Did anything grow in your cultures?  Record the results in Table 1 of the Laboratory Report Form.

2. Complete Tables 2, 3, and 4 of Laboratory Report Form using the descriptive terms found in Figures 1 and 2.   

QUESTIONS:

1. What is meant by a pure culture?

2. Where are microorganisms found?

3. Why must all media and equipment be sterilized?

4. What is agar?

5. What do bacteria in these experiments use as food?

6. How do you know that cloudiness in a broth indicates bacterial growth?

7. Give two reasons why growth may not have occurred.

KEY TERMS:

• agar

• algae

• aseptic technique

• autoclave

• bacteria

• broth

• colony morphology 

• colony

• culture media 

• genus 

• incubator

• microorganisms 

• mold

• petri dish 

• protozoa 

• pure culture 

• slant  

• species 

• sterile

• sterile technique 

• virus

• yeast

SUGGESTED READINGS:

There are special types of culture media known as selective and differential media.  Selective media only allows certain microorganisms to grow on/in it.  Differential media allows different types of microorganisms to grow, but depending on the organism’s metabolism, different microorganisms may appear differently.  Culture media can be selective and/or differential.  This article from the American Society for Microbiology explains why these media are important in helping us identify microorganisms (especially in healthcare!).    

VIDEO CHECK - Can you spot the error??:

Below are three videos,  In each video, what mistake was made?