Microlab Lab Review:

Plate Streak Types

Used to isolate individual bacterial colonies from a mixed culture. Isolation is necessary so that a pure culture can be obtained for identification or further experiments.

Quadrant Streak

Plate divided into 4 sectors

Loop sterilized between streaks

Each streak spreads fewer cells

Final quadrant produces isolated colonies

T-Streak

Plate divided into 3 sections

Streaked in a T pattern

Used when moderate dilution is needed

Zig-Zag Streak

Continuous streak across plate

Not ideal for isolation

Used to simply distribute bacteria

Why streaking works

Each new streak dilutes the bacterial population, allowing single cells to grow into individual colonies.

Parts of the Microscope: Ocular lens

Eyepiece used to observe specimen.

Magnification = 10x

Objective lenses

Objective Magnification

Scanning 4x

Low power 10x

High power 40x

Oil immersion 100x

Stage

Platform where slide sits.

Condenser

Focuses light onto specimen.

Controls brightness and resolution.

Coarse focus knob

Moves stage quickly for initial focus.

Used only with low magnification.

Fine focus knob

Used for precise focusing, especially at high magnification.

Light source

Illuminates specimen.

Oxidation vs Fermentation (O‑F test)

This test determines how bacteria metabolize glucose.

Oxidation

Requires oxygen

Glucose broken down using aerobic respiration

Oxidative Positive

Only open tube turns yellow

Fermentation

Does not require oxygen

Energy produced anaerobically

Fermentation Positive

Both tubes turn yellow

No metabolism

Medium remains green

Obligate Aerobe

Requires oxygen.

Growth pattern: Top of tube only

Example: Pseudomonas

Obligate Anaerobe

Oxygen is toxic.

Growth pattern: Bottom of tube

Example: Clostridium

Facultative Anaerobe

Can grow with or without oxygen.

Growth pattern: Throughout tube but heavier at top

Example: E. coli

Aerotolerant Anaerobe

Does not use oxygen but tolerates it.

Growth pattern: Even growth throughout

Broth Growth Types: Uniform turbidity

Even cloudy growth.

Broth Growth Types: Sediment

Cells settle at bottom.

Broth Growth Types: Pellicle

Growth forms film on surface.

Broth Growth Types: Flocculent

Clumps floating in broth.

Slant Growth

Slants are used to maintain bacterial cultures.

Characteristics to Describe Bacteral Morphology

Shape

Margin

Texture

Pigment

Elevation

Example description:

"Raised, smooth margin, creamy texture, opaque white colonies."

Aperture Number (Numerical Aperture)

Numerical aperture measures the ability of the objective lens to collect light.

Higher NA =

Better Resolution

Where NA is found?

Printed on the objective lens.

Example of NA

40x / 0.65

0.65 = numerical aperture.

No growth in lab experiments

culture dead

inoculation failed

Contamination in experiment

Poor aseptic technic

Incorrect color change in lab

Incorrect incubation

Wrong reagent order

Simple stain

Uses basic dye, positive charge, hence why it stains negatively charged cells.

Simple stain examples

crystal violet

methylene blue

safranin

Purpose of Simple Stain

Observe cell shape, size, arrangement

Heat-fixing may distort cells

Negative stain

uses acidic dye (negatively charged), which is repelled by (negatively charged0 cell walls, leaving clear cells on a dark background

Negative stain example

nigrosin

Negative stain Purpose

Stains background instead of cells

Cells appear clear

no heat-fixing so cells stay in their original state, to see bacteria in their natural size, shape and arrangement

Differential stains

Used to distinguish bacteria. (multiple stains involved)

Differential stains examples

Gram stain

Acid fast stain

Endospore stain

Gram‑positive bacteria

Have 1 lipid layer, thick peptidoglycan, and teichoic acids

Result of Gram + Stain

Retain crystal violet.

Color: Purple

Gram‑negative bacteria

Have 2 lipid layers and thin peptidoglycan and LPS

Gram - Stain

Alcohol removes stain.

Counterstain (safranin) colors them:

Pink

Gram Stain

Crystal violet

Iodine (mordant)

Alcohol (decolorizer)

Safranin (counterstain)

Simple stain Procedure

basic dye

stains bacteria

heat fixed

Result: cells colored

Negative stain Procedure

acidic dye

stains background

no heat fixation

Result: cells appear clear

Advantage: Cells remain true size and shape.

Principle of Acid‑Fast Stain

Used to detect bacteria containing mycolic acid.

Acid Fast Stain

Contain mycolic acid, have a waxy, hydrophobic wall, and that the test is meant for the Mycobacterium genus, including organisms like M. leprae and M. tuberculosis.

What is the principle of Acid-Fast Stain?

Normal water-soluble stains do not penetrate the waxy mycolic acid-rich wall well.

What type of stain is used in Acid-Fast Staining?

Carbolfuchsin, a phenolic stain.

How does steam heat affect the mycolic acid in Acid-Fast Staining?

Steam heat melts the mycolic acid and creates pores that let the stain enter.

Acid fast bacteria: Red

Non acid fast bacteria: Blue

What is in the Cell Membrane

Cell membrane contains:

phospholipids

proteins

enzymes

transport proteins

Function: Controls movement of substances in and out of cell.

Fluid Thioglycollate Medium (FTM)

FTM is used to determine oxygen requirements of bacteria.

The medium contains chemicals that remove oxygen, creating an oxygen gradient.