Microbiology Lab 4

Harpret Guidion

Microbiology 101

Unitek College Fremont

Lab Report #4

Isolation of Individual Colonies

Due April 2, 2017

Abstract

The purpose of this experience is to learn about the two types of culture growth media and the six different types of colony morphology. We will learn to use different types of isolation techniques. This includes the pour plate method, the dilution method, and the streak plate method to prepare cultures.

Hypothesis

This exercise will help determine which method works better than the other type of medias in order to isolate individual colonies.  

Methodology

Exercise 1: Isolation Using the Pour Plate Method

Preparing the solid media:

After disinfecting my work area, I melted the agar tubes in boiling water at 85 degrees Celsius. One petri dish was labeled S. epidermis and poured one half of the contents of a tube of nutrient agar into the S. epidermis petri dish and the other half into the bottom of an unmarked petri dish. Covers were placed and allowed to solidify at room temperature, noting special care to prevent condensation

• Three sterile petri dishes were labeled at the bottom as L. acidophilus #1, #2, and #3.
• Three test tubes were sterilized in boiling water for 5 minutes and then labeled on the bottom as L. acidophilus #1, #2, and #3.
• The hot liquid MRS agar was evenly divided into the three test tubes.
• Using aseptic technique, I inoculated tube #1 with one loop full of the saved L. acidophilus culture and returned into the hot water.
• I inoculated tube #2 with one loop full of the bacteria media mix from tube #1 and returned into the hot water.
• I inoculated tube #3 with one loop full of bacteria media mix from tube #2 and returned into the hot water.
• The contents of L. acidophilus #1, #2, and # 3 test tubes was then poured into the corresponding petri dishes and covered immediately.
• The petri dishes of agar were left to solidify at room temperature and then incubated in an inverted position for 72 hours at 35 degree Celsius.

Results: The petri dishes were examined after 72 hours. The agar hardened and no colonies were observed.

Exercise 2: Isolation and Enumeration by Dilution to Extinction:

• Six prepared agar dishes and six tubes were labeled 10-1 through 10-6.
• Six unmarked agar dishes were labeled 10-1 through 10-6.
• A graduated pipet was used to put 2.25 mL of distilled water in each test tube.
• .25 mL of prepared yeast solution was placed in test tube 10-1. After mixing, .25mL of solution 10-1 was placed in tube 10-2 and was repeated for all tubes.
•  Four drops of each solution were placed in each corresponding agar dish. The contents were swirled and covered and put to incubate for 72 hours.

Results: By using the formula CFU divided by (volume plated (ml) times dilution factor equals CFU/ml original

10-1: TNTC, 10-2: 100 CFU, 10-3: 100 CFU, 10-4: 50 CFU, 10-5: 70 CFU with some grouping, and 10-6: 60 CFU.

Exercise 3: Isolation by the Streak Plate Method

A prepared nutrient agar dish was inoculated with saved S. epidermis from exercise 1 and through aseptic technique by streaking it with a loop in one quadrant. The inoculation loop was disinfected and dragged it through quadrant one to streak quad two. This was repeated twice more. The dish was covered and incubated inverted for 72 hours at 35-37 degrees Celsius.

Results: The S. epidermis agar dish produced raised opaque colonies white in color.

Exercise 4: Stock Cultures

I labeled a tube of nutrient broth S. epidermis Stock Culture and aseptically transferred an S. epidermis colony from the petri dish into the nutrient broth. I then incubated the stock culture for 48 hours. I then stored the culture in a zip bag in the refrigerator for later use.  Next I added a tablespoon of bleach to kill off the other organisms.  After I discarded the contents and disinfected my work area.

Observations and Results

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[pic 3][pic 4]

Analysis

I had fun learning how to isolate bacteria using the pour plate method, the dilution method, and streak plate method.  

Conclusion or Discussion

Overall, I think the lab went well and was pretty straightforward as far as the instructions on how to complete the lab.  The lab exercises for this lab seemed somewhat longer because of having to store and observe the petri dishes after three days

Answer the Questions

Exercise Questions:

A. Define the following:

Enriched Media: An enrichment medium contains some important growth factor         (vitamin, amino acid, blood component, or carbon source) necessary for the         growth of fastidious organisms.

Selective Media: Selective media allow for the selection of particular microorganisms         that may be present in a mixed culture. Selective media usually contain a         component that enhances the growth of the desired organism or inhibits the         growth of competing organisms.

Differential Media: Differential media allow for the separation of organisms based on         some observable change in the appearance of the medium or by an observable         effect on the microbe.

Complex Media: A complex medium is composed of a mixture of proteins and extracts         in which the exact amount of a particular amino acid, sugar, or other nutrient is         not known.

Synthetic Media: In a synthetic medium, the exact amount of pure chemicals used to         formulate the medium is known.

B. Why is it necessary to use a solid agar medium to obtain a pure culture of S. epidermidis?

        To obtain a pure culture it is necessary to separate individual cells of a particular         microbe. This requires the use of a solid medium that provides a surface for the         individual cells to be separated and isolated from the other microbial cells that         might be present in the original sample.