Microbiology and Microbial Techniques
This section covers microbial groups and laboratory methods. The exam tests sterilization, media, streaking, staining, growth kinetics, and viable counting.
Core rules and methods
Bacteria, fungi, algae, protozoa, and viruses differ in cell organization and cultivation. Autoclaving uses steam under pressure at 121 degrees Celsius, 15 psi, for 15-20 minutes. Dry heat uses 160-170 degrees Celsius for about 2 hours. Heat-labile liquids are filter-sterilized. Defined media have known chemicals; complex media use peptone, yeast extract, or blood. Selective media suppress unwanted microbes; differential media reveal colony differences. Streaking dilutes the sample to obtain single colonies. Gram staining uses crystal violet, iodine, alcohol decolourization, and safranin; Gram-positive cells retain purple, Gram-negative cells appear pink. The growth curve has lag, log, stationary, and death phases. Viable counts are reliable when plates have 30-300 colonies.
Worked example
One millilitre of a 10^-5 dilution is plated and 60 colonies appear. CFU/mL = 60 / (10^-5 x 1 mL) = 6 x 10^6. The dilution factor is 10^5, and the plated volume is 1 mL. If only 0.1 mL had been plated, the denominator would be 10^-5 x 0.1, giving 6 x 10^7 CFU/mL.
Traps that cost marks
Autoclaving is not dry heat. A CFU is a colony-forming unit, not necessarily one cell. Colony counts below 30 or above 300 are statistically unreliable. Over-decolourising Gram-positive cells makes them falsely pink. Do not confuse selective and differential media.