Microbial Media, Biochemical Tests, and Antibiotic Resistance: Key Concepts and Experiments

Selective media

Contain chemicals that prevent growth of unwanted bacteria, allowing growth of target bacteria.

Differential media

Contain dyes or indicators to distinguish bacteria based on metabolic or biochemical properties.

Enriched media

Contain nutrients like blood or serum to support fastidious organisms.

Tryptic Soy Agar (TSA)

General purpose, supports many bacteria.

Eosin Methylene Blue (EMB)

Selective (inhibits Gram-positive bacteria) and differential (lactose fermentation indicated by colony colour).

KF Streptococcal Agar

Selective and differential medium targeting streptococci.

Peritrichous flagella

Flagella all around the cell, typically found in Proteus.

Thermal Death Time (TDT)

Time required to kill all bacteria at a specific temperature.

Thermal Death Point (TDP)

The lowest temperature at which all bacteria are killed within 10 minutes.

Facultative anaerobe

Organism that can grow with or without oxygen, such as Escherichia coli.

Obligate anaerobe

Organism that cannot grow in the presence of oxygen, such as Clostridium sp.

Obligate aerobe

Organism that requires oxygen for growth, such as Staphylococcus aureus.

Aerotolerant anaerobe

Organism that does not require oxygen but can tolerate its presence, such as Lactiplantibacillus plantarum.

Cysteine & Sodium Thioglycollate

Reducing agents that lower oxygen to favor anaerobes.

Resazurin dye

Indicator that shows pink color in the presence of oxygen.

Nutrients for growth

Yeast extract & protein provide essential nutrients for microbial growth.

Bacillus megaterium

Spores survive higher temperatures and longer times than E. coli because spores are more heat resistant.

E. coli

Non-spore former that dies faster at elevated temperatures.

Growth Appearance & Color

Describes the visual characteristics of microbial growth in different media.

Endoenzyme activity

Refers to the activity of enzymes that function within the cell.

Catalase Test

Test that determines the presence of catalase enzyme in organisms.

Oxidase Test

Test that determines the presence of oxidase enzyme in organisms.

Osmotic Pressure

Influences microbial growth mechanisms by causing dehydration, enzyme inactivation, and growth inhibition in high salt environments.

Halophiles

Microbes that require high salt concentrations for growth.

S. aureus

Tolerates higher salt concentrations better than E. coli but is inhibited at very high salt (25%).

H. salinarium

A halophile that does not grow at low salt but requires high salt (25%) for growth.

Oligodynamic Action

The ability of small amounts of heavy metals to kill bacteria.

Zone of Inhibition

Size indicates the effectiveness of the metal chloride against bacteria.

NiCl₂

Showed the largest zones of inhibition for both E. coli (5 mm) and S. aureus (3 mm), indicating the strongest antibacterial effect.

PbCl₂

Had no inhibition zones on either bacteria, meaning it had no noticeable antimicrobial effect at the concentration tested.

BaCl₂

Had minimal or no effect, especially on S. aureus.

MnCl₂

Had minimal or no effect, especially on S. aureus.

Gram-negative

Bacteria like E. coli that appeared generally more susceptible to the metals tested.

Gram-positive

Bacteria like S. aureus that showed smaller or no zones of inhibition.

Disinfectant

Chemical agents used to kill or inhibit microorganisms on inanimate objects.

Antiseptic

Chemical agent safe to use on living tissue that kills or inhibits microbes.

Cidal

The agent kills microbes, preventing regrowth after removal.

Bacteriostatic agents

Agent inhibits microbial growth temporarily; microbes may regrow after the agent is removed.

Static

Agent inhibits microbial growth temporarily; microbes may regrow after the agent is removed.

Chemical Agent

Substances used to measure and compare the susceptibility of microbes by their zones of inhibition.

Lysol

An example of a disinfectant.

Dettol

An example of an antiseptic.

Iodine

An example of an antiseptic.

Bactericidal agents

Agents that kill microbes, preventing regrowth after removal.

5% Lysol

A disinfectant concentration used in testing.

10% Dettol

A disinfectant concentration used in testing.

2% Iodine

A disinfectant concentration known for strong antimicrobial activity.

Scope

A disinfectant whose effectiveness might be weaker or unknown.

Gram-positive bacteria

Bacteria with a thick peptidoglycan layer and no outer membrane.

Gram-negative bacteria

Bacteria with an outer membrane that can block or slow chemical penetration.

Antibiotic

An antimicrobial agent produced by microorganisms that selectively inhibit or kill other microbes.

Disc diffusion method

A method where antibiotic discs diffuse into agar inoculated with bacteria to measure sensitivity.

Tetracycline

An antibiotic that prevents tRNA binding to 70S ribosomes.

Penicillin G

An antibiotic that inhibits peptidoglycan synthesis.

Erythromycin

An antibiotic that binds 23S rRNA in the 50S ribosomal subunit.

Polymyxin B

An antibiotic that alters membrane permeability by binding phospholipids.

30 mcg

The concentration of Tetracycline used in the experiment.

10 units

The concentration of Penicillin G used in the experiment.

15 mcg

The concentration of Erythromycin used in the experiment.

300 units

The concentration of Polymyxin B used in the experiment.

9mm

The zone diameter for S. aureus when treated with Tetracycline.

16mm

The zone diameter for E. coli when treated with Tetracycline.

2mm

The zone diameter for S. aureus when treated with Penicillin G.

17mm

The zone diameter for E. coli when treated with Penicillin G.

4mm

The zone diameter for S. aureus when treated with Erythromycin.

0mm

The zone diameter for E. coli when treated with Erythromycin.

15mm

The zone diameter for E. coli when treated with Polymyxin B.

Antibiotics

Naturally produced chemical substances from microorganisms that inhibit or kill other microbes.

Antimicrobial drugs

Include antibiotics plus synthetic agents (like sulfonamides) that act against microbes.

Broad spectrum antibiotics

Effective against a wide variety of bacteria (both Gram-positive and Gram-negative), e.g., tetracycline, erythromycin.

Narrow spectrum antibiotics

Effective against a specific group, e.g., penicillin primarily against Gram-positive bacteria.

Factors influencing zone size

Rate of diffusion of antibiotic in agar, inoculum size and density, growth rate and susceptibility of the microorganism, incubation time and temperature.

Mutations in bacteria

Arise due to genetic changes—either spontaneously during DNA replication or induced by mutagenic agents (mutagens) such as UV rays, X-rays, nitrous acid, alkylating agents, or base analogues.

Antibiotic-resistant mutants

Survive and grow even in the presence of antibiotics that inhibit or kill the wild-type bacteria.

Penicillin gradient plate

The plate has a gradient of penicillin concentration, from low (L) on one side to high (H) on the other side.

Expected Observations in penicillin gradient experiment

Numerous colonies grow on the low concentration side. As penicillin concentration increases across the plate, fewer colonies grow. Resistant colonies (mutants) appear on medium and high concentration zones.

Confirmation of resistance

Isolate and culture it in the presence of penicillin at the same or higher concentration to confirm growth. Perform sensitivity testing (zone of inhibition tests).

Horizontal gene transfer

Mechanism by which bacteria gain resistance besides mutation, involving plasmids and transposons.

Natural selection under antibiotic pressure

A process that contributes to the development of antibiotic resistance in bacteria.

Penicillin resistance commonality

Resistant mutants have a selective advantage and multiply in presence of penicillin; also, gene transfer spreads resistance.