Exercise 3 - Culturing of Microorganisms

Exercise 3 - Learning Objectives:

Following this exercise the student should be able to:

  1. explain the principles behind the use of solid and broth media for culturing.
  2. perform an environmental culture using the appropriate tools and techniques.
  3. identify and describe three different colony morphologies grown from their specimen.
  4. interpret the significance of growth in a broth culture.
  5. evaluate the purity of a culture.

Bacteria are invisible to the naked eye. In order to study them we use two significant techniques: 1) microscopy, in conjunction with staining to make them visible and 2) culturing bacteria so that there will be large enough numbers to observe masses of bacteria with the naked eye. A single bacterium multiplies by binary fission to produce a characteristic mass of genetically identical bacteria. This mass is called a colony and is considered to be genetically pure. The shape, consistency, color, margin (border), and elevation of the colony are as characteristic of a species of bacteria, as the shape size and color of a fish is to a species.

When grown in liquid media (broth culture) bacteria will cause the tube to look cloudy in the region where growth is heaviest. Some bacteria form a pellicle (a thick layer) on the surface of the fluid, others form cotton ball shapes, and some appear to have sunk to the bottom of the tube as if sticking to the bottom. This growth relates to the bacteria's oxygen requirements. The microbiologist cannot pick out individual bacterial types from a broth. In order to separate and isolate different bacterial types they are grown on solid media. This media has all the required nutrients, as the broth culture had, but a solidifying agent, called agar, is added to the mixture. Solid media allows many observations to be made about the colonial morphology of different bacteria. We use solid media in tubes (agar deeps), in tubes that have been tipped and allowed to solidify with a surface for growth (agar slant), and in plates called Petri dishes.  

Agar slants (in tubes) are useful for maintaining bacterial growth over long periods of time without drying. Petri dishes allow observation, easy access, and isolation; but are prone to environmental contamination and desiccation.

 

Investigative Assignment

Using the transport culture swab, select a location that you hypothesize may contain many invisible microbes. Just before class, open the paper cover and using aseptic technique* roll and rub the cotton swab on the area you want to culture. Place it back in the sterile** plastic sleeve. Label the culture source and break the ampule at the bottom of the container to release the transport medium that will nourish the microorganisms until you subculture the specimen.

 

 

                                                                                                             

              

                       Selected surface for culturing

 *Aseptic technique means free of contaminating organisms other than those you are investigating. For example, you would not want your hand microbes to contaminate the swab.

**Sterile means free from all life forms.

Lab Period 1-

Materials for each individual:                          

PPG (personal protective gear)
1 inoculated culturette swab
1 Tryptic Soy Agar (TSA) plate
1 Tryptic Soy Agar (TSA)  slant
1 nutrient broth
transfer loop
gloves

Materials for each lab team:
Test tube rack
small beaker with disinfectant
incinerator
sharpie

Method:

The streak plate method is used to isolate individual colonies of bacteria for observation and identification. After 24 hours of growth the final product should look like this picture.

This is tricky; the bacteria must be dispersed while they are still invisible to your eyes.

Check out this power point explanation for Isolation Streaking.


Proceed as follows placing a check in the box at the end of each step after it is completed:

  1.  Plug in the incinerator, put on PPG.

  2.  Label the tubes and plate with your initials, the culture source, and the date. Read the Atlas on inoculating a streak plate (page 9).

  3. Remove the swab from the plastic container. Open the Petri dish placing the lid upside down on a surface thereby preventing contamination of the lid and lab top. Without contacting the swab to any other surfaces, roll the swab across quadrant number 1 of the agar plate, the initial surface to be cultured.  Recover and replace the Petri dish to limit contamination from the air. Replace the swab in the plastic container.

  4.  Sterilize the loop, by holding it in the incinerator for 10 seconds. Remove the loop, hold it as it cools. (This is composed of nichrome wire, a rapidly heating and cooling type of metal.)

  5. Carefully open the Petri dish, placing the lid upside down once again. Touch the surface of the agar to gauge its durability. You want to rub the bacteria across the agar without gouging it. Rub the loop gently over the surface of the medium at the very end of quadrant one and through quadrant 2 as depicted in the atlas.

  6.       Sterilize the loop, allow it to cool. Streak quadrant 3 as shown in the diagram.

  7.       Sterilize the loop, allow it to cool. Make the final broad strokes through quadrant 4 as shown in the diagram. Sterilize the loop and allow it to cool.

  8.    Inoculate the broth culture. Using the culturettes again, aseptically remove the swab. Open the top of the nutrient broth; place the top of the tube near the incinerator (5 seconds). Insert the swab in the broth. Reheat the mouth of the tube. Close the tube and replace the swab.

  9.    Inoculate the agar slant. Using the culturettes again, aseptically remove the swab. Open the top of the agar slant; place the top of the tube near the incinerator (5 seconds). Insert the swab and roll it up the slant. Reheat the mouth of the tube. Close the tube and replace the swab back in the plastic sleeve.

  10. Label everything. Incubate the plate (upside down). Place the agar slant and broth culture in a test tube rack; put the rack and plate in the 37C incubator. Check the temperature on the incubator. Be sure the doors are completely closed to assure the proper temperature is maintained.

  11.  Turn off the incinerator.

  12.  Place the contaminated material in the appropriate location at the biohazard table (e.g. used culturettes and gloves in the biohazard can with red bag).

  13. Return other materials and equipment to the appropriate location, including the cooled incinerator.

  14.  Wipe down the table top with disinfectant.

  Lab Period 2 -

Materials for each individual:                           Materials for each lab team:

PPG (personal protective gear)
3 Tryptic Soy Agar (TSA) plate
transfer loop
gloves
small beaker with disinfectant
incinerator
sharpie

 

  1. Plug in the incinerator.
  1. Put on your PPG and collect your plates for observation.
  1. Examine the various colony morphologies present on your plate. * DO NOT ever open any plate that has a white fuzzy growth on it – in Bakersfield this could be the Valley Fever Fungus. Call the instructor if anything looks fuzzy.
  1. Select 3 different types of colonies. Number them using your sharpie and writing on the bottom of the plate (not the lid – it moves).
  2. Review in the Atlas (pages 1-4) terminology for the macroscopic observations of colonies on solid media. Refer to the table below for examples of typical terms used to describe colonial morphology. Practice using these terms to describe colonies to your lab partners.

  For example this colony could be described as white, raised, rhizoid, opaque. It is hard to tell in the picture whether it is dry or moist.

These colonies would be described as spreading, irregular, moist, and gray.

 

 

 

 

Colonies of Swarming Proteus mirabilis© Shapiro, author. Licensed for use, ASM MicrobeLibrary (linked to http://www.microbelibrary.org)

 

 

 

Common Descriptions for Colony Morphology

Colony Size Size of colonies correlates to some species

Punctiform

Very small dots           .:.:

Small Small colonies are relative, of course, but colonies with a diameter of 1-2 mm would be considered small
Large colonies Regardless of the shape, colonies ranging over 8 mm would be considered large
Borders The outer edge of the colony

Circular

Round colonies with even borders

 

Filamentous

Fibrous looking, with stringy extensions

                                                                                                         

Irregular

Extending unevenly from the center with asymmetrical borders

 

Loboid

Border of colony appears to extend out in lobes

 

Rhizoid

The border of the colony appears to extend out like tree roots - see picture and description above

Spindle

The colony is shaped long and thin like a spindle

 

Spreading or swarming

Edges of the colony appear wavy                See picture above

Elevation

The elevation of the colony refers to the height or depth of the colony compared to the plane of the agar

Flat

Colony seems even with the surface of the agar

Raised

Colony appears as a mound on the agar

Umbonate

Slightly raised in the center     

 

Pitted or concave

Depressed into the agar

Texture The texture of the colony relates to it moistness or dryness, whether you can see though it or it is opaque

Shiny or Wet

Colony appears shiny and moist

Mucoid

Colony appears covered with mucous

Dry

Colony appears dry and sometimes folded

Opaque

Colony is solid, an object behind the colony can not be seen

Translucent

A colony that appears to allow you to see through it

Color Most colonies have some color - many are just shades of white or grey, but the color is an important characteristic and tool in identification

Colored Colonies

Some colonies have characteristic colors other than the color of the agar such as: Orange, Yellow, White, Pink, Black, Red

Translucent As described above, this colony would appear clear, like glass, or a drop of water

                                                                                                         
Lab Report for Exercise #3

 

Name _________________________ Culture Source________________________

 

   1. Select 2 or 3 different colonies from your culture.  Draw a sketch of each colony below. Describe the colony on the chart below using the descriptive terminology on the previous page. You may use the colony counter to get a better view.

 

 

Colony

 

Sketch

 

Description

 

 

#1

 

 

 

 

 

 

 

 

#2

 

 

 

 

 

 

#3

 

 

 

 

 

 

 

2. Subculture these organisms by transferring each of these bacteria to a new plate using the isolation technique described in the Atlas (see page 9). These colonies will be used to practice staining next lab hour.

Original environmental mixed culture

 

   

          Streak for isolation
           

            

Three pure cultures                                     

 

 

3. Observe the growth in your broth culture. Draw a picture of the tube in the box above indicating bacterial growth. Circle the type oxygen requirement this growth indicates:

 strict aerobe          microaerophile         facultative anaerobe         strict anaerobe      mixed growth & requirements

 

 4.      List the advantages and disadvantages of a broth culture compared to a solid culture.

 

 

 5.      Why is the plate incubated upside down?

 

 

 6. What would occur if the lid of the Petri dish was placed right side down on a tabletop?

   

7. You are asked to use a culturette to collect a wound specimen. Would you inoculate the specimen on to solid agar plate, a slant, or into a broth culture? Explain why.

 

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Bakersfield College | Kern Community College District | Dr. Janet Fulks
1801 Panorama Dr. - Bakersfield, CA 93305 - (661)395-4381
Date last updated 07/21/2011
©Janet Fulks