Is macconkey agar a defined or undefined medium

Microbiology Lab

Lab 5- Culture Media and Contamination
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 4.5 and 4.6) (Nester- McGraw Hill)

· Lab Manual by Dr. Su, pages 39-44 (optional)

· Link: Cultivation of Bacteria-Media, Virtual Edge Experiment- 3B

Cultivation of bacteria in the laboratory- Culture Media
Read the information from textbook from chapter 4 review sections 4.5 (pages 103-105) and 4.6 (pages 106-107) to answer the following questions.

Natural Growth requirements of bacteria
1. List the major elements required for bacterial growth along with their function.

2. List the trace elements required for bacterial growth along with their function.

3. What is a “limiting” nutrient?

4. What are growth factors?

5. What is meant by fastidious bacteria? How are these used in research and industry?

6. Define the following and give one example of an organism that belongs to each category:

a. Autotroph

b. Heterotroph

c. Photoautotroph

d. Photoheterotroph

e. Chemolithoautotroph

f. Chemoheterotroph

Laboratory Growth of Bacteria
· Media can be SELECTIVE; allowing only certain types of microorganisms to grow on it. The addition of any antibiotic to a media makes it selective (i.e. only microorganisms that are resistant to that antibiotic would be able to grow in/on that media).

· Media can be DIFFERENTIAL; allowing different types of microorganisms to grow on it, but depending on the organism’s metabolism, different microorganisms may appear differently

· A culture medium can be both selective and differential.

Culture medium

Principle

Expected results

EMB

(Selective & Differential)

Selective culture medium for Gram-negative bacteria (selects against Gram-positive bacteria) and is commonly used for the isolation and differentiation of coliforms and fecal coliforms.

Eosin and methylene blue inhibit Gram-positive and fastidious Gram-negative bacteria.

EMB agar (Eosin-Methylene Blue agar) - Sharebiology

· Gram-negative bacteria that ferment the lactose produce acid which turns the colonies dark purple as the acid acts upon the dyes.

· In addition, certain lactose-fermenting bacteria produce flat, dark colonies with a green metallic sheen due to rapid fermentation of lactose & production of strong acids.

· Other lactose fermenters produce larger, mucoid colonies, often purple only in their center.

Buy Emb Agar | Levine Eosin Methylene Blue | Tm 371 | Tm Media | Microbiology, Methylene blue, Microbiology lab

MSA

The medium contains 7.5% concentration of sodium chloride (high salt) which results in the partial or complete inhibition of bacterial organisms other than staphylococci. Mannitol is the fermentable carbohydrate source, fermentation of which leads to acid production. The pH indicator- phenol red turns yellow at acidic pH.

Results on Mannitol Salt Agar (MSA)

Staphylococcus aureus and Staphylococcus epidermidis on Mannitol Salt Agar. Mannitol salt agar composition. Positive and negative result on mannitol salt agar.

Blood Agar

(Differential)

The addition of blood makes the medium more nutritious by providing additional growth factors required by fastidious organisms. It also helps in visualizing the hemolytic reactions. Hemolysis, the ability to lyse erythrocytes, is carried out by enzymes called hemolysins. Different bacterial species have different classes of hemolysisns- . (Hemolytic reactions also depend on the animal blood used).

· Alpha hemolysis: Colonies showing partial red blood cell breakdown demonstrates alpha-hemolysis, shown by Streptococcus pneumoniae

· Beta hemolysis: Colonies showing complete red cell breakdown demonstrate beta-hemolysis, shown by Staphylococcus aureus

· Gamma hemolysis: Colonies not showing red blood cell breakdown demonstrates gamma-hemolysis, shown by Enterococcus faecalis, Klebsiella pneumoniae

Blood Agar- Composition, Principle, Preparation and Uses

McConkey Agar

(Selective & Differential)

Isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria, particularly members of the family Enterobacteriaceae and the genus Pseudomonas.

· The inclusion of crystal violet and bile salts in the media prevent the growth of gram-positive bacteria and fastidious Gram negative bacteria.

· Some Gram-negative enteric bacteria tolerate bile partly due to the resistant outer membrane.

· Gram-negative bacteria growing on the media are differentiated by their ability to ferment the sugar sorbitol.

· Bacteria that ferment sorbitol cause the pH of the media to drop and the resultant change in pH is detected by neutral red, which is red in color at pH's below 6.8. As the pH drops, neutral red is absorbed by the bacteria, which appear as bright pink to red colonies on the agar.

· Gram-negative bacteria that grow on MacConkey agar but do not ferment sorbitol appear colorless on the medium and the agar surrounding the bacteria remains relatively transparent

Colony Morphology on MacConkey Agar

· Link: Cultivation of Bacteria-Media, Virtual Edge Experiment- 3B and the textbook chapter 4 (sections 4.5 and 4.6) (Nester- McGraw Hill) to answer the following questions:

7. What is a complex (or undefined) medium? Give one example.

8. Describe a situation that would require you to use complex medium in the lab.

9. What is a chemically defined (or defined) medium? Give one example.

10. Describe a situation that would require you to use chemically defined (or defined) medium in the lab.

11. What is selective media?

12. Describe a situation that would require you to use selective medium in the lab.

13. What is differential media?

14. Describe a situation that would require you to use differential medium in the lab.

15. What are the components of the following media?

a. Nutrient broth

b. GSA

c. TSA

d. EMB

e. MSA

f. Blood agar

g. McConkey agar

16. Out of types of media listed above in question 15 which ones are

a. Complex

b. Defined

c. Selective

d. Differential

e. Selective and Differential

17. Observe the pictures and answer questions:

a. The plate shown below has E. coli growing in the yellow area. True OR False. Explain your answer.

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b. The plate shown below has E. coli growing in the A area. True OR False. Explain your answer.

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c. If a drinking water supply is contaminated with fecal bacteria like E. coli, which of the above tests would you use to confirm?

d. Streptococcus pyogenes causes strep throat. Which of the above tests would you use to confirm an infection with Streptococcus pyogenes? What results of this test would conform if one has strep throat?

e. Based on the results on the McConke’s agar plate below, which letter represents

MacConkey Agar, 500 g | Media for Salmonella & Shigella | Anhydrous Nutrient Media for Microbiology | Microbiology | Life Science | Carl Roth - International

i. Lactose fermenting bacteria like E. coli

ii. Non-lactose fermenting bacteria like Salmonella typhimurium

Cultivation of bacteria in the laboratory- Contamination
Hers is an experiment shown on the Virtual Edge- 1A that we complete in the lab.

After viewing the video and looking at the results you will realize the following:

Our fingers and overall skin carry germs so any time we touch our skin and then touch anything else including food or an inanimate object, we transfer germs. These germs could be bacteria, viruses or fungi.

You don’t get sick every day which actually means not all germs make you sick. Some do but at what extent you will get sick will depend on your immune system too.

Does this mean you have to take extreme care about sanitizing everything you touch? Not really. Exposing to many germs is necessary to prime our immune system! (Under current “COVID” conditions you have to!).

OPTIONAL: Such experiment can be performed at home by

· cutting equal size pieces of apple or potatoes with a clean (sterile) knife in clean containers

· rubbing each one with clean/sterile cotton swabs with different things including keys, cell phone, washed and unwashed fingers, lemon juice (as a preservative)

· covering each container with a piece of clean foil

· keeping in a warm place (room temperature- around 25oC or 77 oF) for 3-5 days observing twice a day.

If you happen to do this experiment at home, share pictures with the class. (Unfortunately, under current difficult conditions, I won’t advise anyone to specially go out and buy anything. After completion of the experiment, the used pieces should be disposed off into trash by wrapping in a plastic bag. None of this should be consumed by anyone including humans and pets. Be extra careful with children and pets.