12.3D Microbiology

Question 1

Sterilization

Answer 1

refers to any process that eliminates, removes, kills, or deactivates all forms of life and other biological agents present in a specified region, or in a compound such as biological culture media

Question 2

Sterilization methods

Answer 2

• Heat (moist heating, draining, burning and dry heat),
• Chemicals (sodium hypercholoride, alcohol, detergents),
• Irradiation,
• High pressure (autoclaving),
• Filtration

Question 3

Aseptic

Answer 3

free of or using methods to keep free of pathological microorganisms

Question 4

Aseptic technique

Answer 4

means using practices and procedures to prevent contamination from pathogens

It involves applying the strictest rules to minimize the risk of infection

Question 5

Antiseptic

Answer 5

is a substance that inhibits the growth and development of microorganisms

Question 6

Inoculation

Answer 6

is the process of introducing microorganisms into a growing medium which is suitable for their growth

Question 7

Incubation

Answer 7

the process of allowing microorganisms to grow in a medium by providing necessary growth conditions

Question 8

Microbial growth

Answer 8

• lag phase - no rapid growth or multiplication. They start adjusting to the new environment, stabilizing
• log phase - optimal growth rate & multiplication
• stationary phase - growth and dying rates equal, growth curve is a straight line parallel to the x-axis
• death phase - death rate exceeds growth rate. After several days, microbial growth ceases, leaving behind a dead culture.

Question 9

Gram staining

Answer 9

• Gram-positive bacteria (thick layer of peptidoglycan-90% of the cell wall)- stains PURPLE.

• Gram-negative bacteria (thin layer of peptidoglycan-10% of cell wall and high lipid content) – stains RED/PINK.

Question 10

Gram Staining Procedure

Answer 10

Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram’s iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.

4. Flood slide with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add drop by drop to slide until decolorizing agent running from the slide runs clear.
5. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
6. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.
7. Observe the results of the staining procedure under oil immersion (100x) using a Bright field microscope.