Bacillus and Mycobacterium

Question 1

Bacillus:
1) Gram stain
2) Morphology
3) Identifying traits
4) Unique behaviors/characteristics
5) What diseases is the organism known for/what diseases can the organism cause?

Answer 1

1) Positive
2) Rods
3) All species are catalase positive
4) Form endospores under stressful conditions
5) B. anthrax: Anthrax; B. cereus: food poisoning; B. subtillus: considered nonpathogenic, probiotic use.

Question 2

How does KL Agar work? What are the important components of the media? Why did we use it?

Answer 2

KL Agar determines the sugar fermentation patterns of Bacillus, Corynebacterium, and strict anaerobes. The media contains ingredients to enhance the growth of Bacillus (which ingredients????? IDK)

Question 3

What does Nutrient Gelatin Deep test for? What does a positive result look like?

Answer 3

Nutrient Gelatin Deep media tests for the enzyme gelatinase which hydrolyzes gelatin. For a positive result, the hard, stiff media will liquefy at room temperature

Question 4

What are steps of Spore Staining? Why did we stain each organism the way we did? What were the stains used and what do they stain? Know and understand how to interpret a spore stain. Know characteristics of organisms.

Answer 4

1. Prepare smears of organisms to be tested for endospores
2. Heat fix the smears
3. Cover the smears with a piece of absorbent papercut to fit the slide and place the slide on a stand
4. Saturate the paper with malachite green and heat slide over steam for 5 minutes
5. As the paper begins to dry add a drop or two of malachite green to keep it moist, but don’t add so much at one time that the temperature is appreciably reduced. DO NOT OVERHEAT. The process is steaming and not baking
6. Drain the slide and counterstain for 45 seconds with safranin
7. Wash, blot, and examine
8. The vegetative cells will appear read, and the spores will appear green

We use spore staining to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers. The malachite green stain stains the spores green and the safranin stain the vegetative cell walls red.

Question 5

Mycobacterium:
1) What is special about this organism’s cell wall?
2) What are the unique characteristics about the organism’s growth/resistances
3) What are medically relevant mycobacterium that we covered and what disease do they cause?

Answer 5

1) The cell wall is thick, waxy, and rich in mycolic acids
2) It is fastidious and slow growing (7+ days in incubation)
3) M. tuberculosis and M. bovis: tuberculosis in humans and animals respectively
M. leprae: leprosy
M. fortuitum: skin and bone/joint infections in healthy and immunocompromised individuals
M. smegmatis: usually skin infection
M. phlei: septic arthritis

Question 6

What are NTM? What differentiates them from other mycobacterium?

Answer 6

NTM are nontuberculosis mycobacterium and they are differentiable because they do not cause tuberculosis

Question 7

Why is the Lowenstein-Jenson Agar important? What does it contain that makes it important?

Answer 7

Lowenstein-Jenson Agar is important because it selects for slow growing mycobacterium sp. It contains malachite green which makes it important and also selects for mycobacterium.

Question 8

Why did we use Middlebrook-Cohn 7H10 Agar? What does it contain that makes it important?

Answer 8

We used Middlebrrok-Cohn 7H10 Agar to determine the antimicrobial susceptibility of an organism. It contains inhibitors to prevent contaminants from growing, the inhibitor is malachite green.

Question 9

What does Arylsulfatase Broth test for? What is biochemical mechanism of the media? What is a positive result?

Answer 9

Arylsulfatase broth tests for rapid growers. The media splits phenolphthalein from tripotassium phenolphthalein. A positive result is a pink-red color

Question 10

What does Tween 80 Broth test for? What is the biochemical mechanism of this media? What is a positive result?

Answer 10

Tween 80 broth tests if an organism can produce lipases that hydrolyze tween 80. Biochemically, the lipases hydrolyze tween 80. A positive result is a red color indicating that there is no tween 80 left in the media.

Question 11

What does Urea Test test for? What is the biochemical mechanism of this media? What is a positive result?

Answer 11

The Urea Test tests for the production of urease. Urease will breakdown urea and turn it into ammonia, alkalinizing the media, changing the pH and thus the color due to a pH indicator. A positive result is a red color

Question 12

Why did we use MacConkey Agar Plate for this lab? What is the biochemical mechanism of this media?

Answer 12

We used MacConkey Agar Plates in this lab because they help to differentiate potentially pathogenic from non-pathogenic mycobacterium. Lactose monohydrate is the fermentable source of carbohydrate. This media is selective due to the presence of crystal violet and bile salts, which are inhibitory to most species of gram-positive bacteria. Sodium chloride maintains the osmotic balance in the medium.

Question 13

Acid fast stain (Ziehl-Neelsen Stain):
1) Understand the stains we did
2) Understand why we stained the organisms the way we did
3) Understand what the stain should look like
4) Know how to interpret an acid fast stain

Answer 13

1) Carbol fushin and methylene blue were used….IDK WHY
2) The steam bath is used because it allows the stains to penetrate the thick cell wall of the mycobacterium
3) A positive stain will have red colored cell walls while a neative stain will have blue colored cell walls
4) Acid fast bacteria will be red while non acid fast bacteria will be blue.