Isolation of pure culture by streak plate method

Microbiology Lab

-Lab 6- Pure Culture, Bacterial Enumeration
Directions:
Answer following questions after reading the information and watching the video from the link below. Use color RED or BLUE for your answers. Submit the completed document on eCampus for grading. Refer to

· the textbook chapter 4 (sections 4.3 and 4.7) (Nester- McGraw Hill)

· Lab Manual by Dr. Su, pages 45-50 (optional)

· Link: Cultivation of Bacteria-Streak plate, Virtual Edge Experiment- 3A

Cultivation of bacteria in the laboratory- Streak plate
Read the information from textbook from chapter 4 review sections 4.3 (pages 96-97)) to answer the following questions.

Pure Culture
1. What is a pure culture?

This is the population of multicellular organisms or cells that grow in the absence of other species or types. They originate from one cell or a single organism, in this case the cells are the genetic clones of each other.

2. What is the need for a pure culture of bacteria?

It facilitates the application of recombinant DNA technology through the isolation of clones. Pure culture tends to react differently in isolation as opposed to when it is combined with other species.

3. How many bacteria are required to start a colony?

One bacterium

Streak Plate Method:
Links: Triple streak, Quadrant Streak

4. What is the purpose of sterilizing the loop in between streaks in this method?

Sterilization dilutes out the sample placed in the initial region of the agar surface.

5. Is this method useful for quantifying bacterial cells in the original culture? Yes Or No- explain your answer:

Yes, the method can be used in quantifying the cells in a culture through plating a known volume of the cell culture on a petri dish with a growth medium.

6. Study the picture below: Which step showed isolated colonies and why?

Isolated colonies are formed once the lone bacterial cells divide and form thousands of new bacterial cells after media inoculation.

Identification of Bacteria - ppt download

7. The picture from question number 7 shows 3 types bacterial species appearing on red, white and yellow colonies. What does this tell you about the original culture?

Colony morphology is a unique way of identifying bacteria.

Pour Plate and Spread Plate methods
Both these methods are “quantitative” meaning you can find out how many bacterial cells you are working with.

· Watch this video as well: Serial Dilution

· Results: Notice how colonies are counted, why are some colonies embedded in the agar in the pour plate method.

· Link: Enumeration of Bacteria, Virtual Edge Experiment- 7B and the textbook chapter 4 (sections 4.5 and 4.6) (Nester- McGraw Hill) to answer the following questions:

· The video in the above link shows “Spread Plate” technique which makes used of agar plates to spread the diluted samples.

· In the Pour Plate technique, liquefied agar cooled to 45-50oC is used instead of a ready agar plate.

8. What is the difference in the spread plate and pour plate method?

In pour plate a known volume of the sample is mixed with agar and poured into a plate while in spread plate a known volume of the sample is spread on the surface of the agar medium.

9. Which of the two methods, spread plate and pour plate has better separation of bacterial colonies?

Pour plate method

10. Why is it necessary to cool down agar to 45-50oC?

To prevent condensation from forming on the lid. It ensures the bacteria does not die when being transferred to the agar and to ensure the agar does not solid.

11. Give two scenarios where the number of bacterial cells may be important.

Persister cells are medically important. The milk-souring bacterial genus lactobacillus in making yoghurt.

12. What is CFU?

This is a colony forming unit used to estimate the number of viable bacteria or fungal cells within a sample.

Serial dilutions are done as shown in the picture below:
4: Dilution Worksheet and Problems - Biology LibreTexts

Sample CFU problems:
One ml of a bacterial culture was pipetted into a 9 ml dilution blank. One-tenth ml of this dilution was pipetted into a 9.9 ml dilution blank. From this dilution, one-tenth ml was plated with 25 ml of culture medium. 220 colonies arose after incubation. How many colony-forming units were present per ml of the original culture?

http://www.jlindquist.net/generalmicro/DTimages/ps1a.jpg

Working through the problem with the formulas:
Formula-1: dilution made X amount inoculated = "plated dilution"

1/10 X 1/100 X 1/10 = 1/10,000 or 10–4

Formula-2: dilution factor X number of colonies = number of CFUs/ml

104 X 220 = 2.2 X 106

As we understand the so-called "plated dilution" to represent the actual amount of undiluted sample being tested, we can look at the problem (and similar problems) like this: If 220 colonies arose from plating (the equivalent of) 10–4 ml of the culture, then (proportionally) there would have been 220 X 104 which is same as 2.2 X 106 CFUs per one ml of the original, undiluted culture . This is the reasoning behind the second of the two dilution formulas.

The dilution factor is the inverse of the plated dilution and is meant to inflate the colony count to give the number of CFUs that were in one ml of the original, undiluted sample at the time it was plated out.

Now solve problems 13-14
13. Ten ml of spring water were added to a petri dish to which 40 ml of melted Plate Count Agar were added. After incubation, 35 colonies arose on the plate. What was the count of CFUs per ml of the spring water?

The dilution facor is 10-1. The plated solution represents that actual amount of sample being tested i.e. 10 ml. the inverse of the plated dilution i.e. dilution factor would be 10-1

14. You have obtained a pond water sample and wish to determine the concentration of bacteria which are gram-negative and lactose-fermenting. After making two 1/100 dilutions, you plate 0.1 ml of the second dilution onto each of two plates of MacConkey Agar. After appropriate incubation, you find that one plate contains 155 red colonies and 45 white colonies, and the other plate contains 160 red colonies and 35 white colonies. What was the concentration of gram-negative, lactose-fermenting CFUs per ml of the pond water sample?

Only gram-negative bacteria should grow on MacConkey agar. The red colonies are lactose-fermenters. The average number of colonies of lactose-fermenting gram-negative bacteria is 157.5.

dilutions made X amount inoculated = "plated dilution"

1/100 X 1/100 X 1/10 = 1/105 or 10–5

dilution factor X no. colonies = no. CFUs/ml

105 X 157.5 = 1.6 X 107