Microbiology Lab Techniques

Gram Staining

• Gram staining is used to classify bacteria as either gram-negative (pink or red) or gram-positive (blue/purple).
• Gram-positive bacteria have a thick peptidoglycan layer, which retains the crystal violet stain.
• Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane made of lipopolysaccharides.
• The primary stain, crystal violet, stains all bacteria purple.
• Gram's iodine acts as a mordant, forming a crystal-violet-iodine complex.
• Ethanol is used as a decolorizing agent, washing the crystal violet-iodine complex out of gram-negative bacteria.
• Safranin is applied as a counterstain to color the decolorized gram-negative bacteria red.
• Older or dead bacterial cells may not retain the crystal violet-iodine complex.
• Overheating during heat fixing can rupture cell walls.
• Smears that are too thick may prevent reagents from reaching all cells.
• Leaving dye on for too long can stain the glass slide, obscuring the bacteria.
• Picking up agar during the procedure can make it difficult to see bacteria.

Streak Plate Method

• The streak plate method is used to grow isolated colonies of bacteria on agar plates.
• The purpose is to dilute bacterial cells, allowing them to grow into pure colonies, each derived from a single cell.
• The loop needs to be flamed between streaks to maintain purity.
• A needle is used to inoculate agar deeps, not broth cultures.

Culture Media

• Semisolid agar deeps are used to determine bacterial motility.
• Agar slants are used to store pure cultures.
• Broth cultures are used to grow large numbers of bacteria.
• Agar deeps are used to test bacterial oxygen requirements.

Microscopy

• When using oil immersion lens, oil should touch the lens and the slide.
• Agar slants are intended for pure cultures, so the organism should have only one type of growth.
• Eukaryotes have membrane-bound organelles that can be seen under low or high-dry powered objectives.

Hand-Washing Experiment

• Transient bacteria and normal flora are removed by hand-washing.
• Normal flora is most likely to remain after hand-washing.
• Independent variables: Handwashing time and the use of soap.
• Dependent variable: Number of bacterial colonies.

Bacterial Colony Differentiation

• Bacterial colonies can be distinguished by their form, margin, surface, color, size, and translucence/opacity.

Controls in Experiments

• A control is a standard of comparison for verifying results, ensuring that the outcome is a result of the tested variable.

Structural Stains

• Structural stains identify and enable study of bacterial structures.
• These are a type of differential stain.
• They are useful for observing bacterial endospores, capsules, and flagella.
• Structural stains can help ID unknown organisms.

Endospores

• Form in gram-positive bacteria when nutrients or water are unavailable.
• They are non-metabolizing, non-reproducing, and highly resistant to heat and chemicals.
• They allow bacteria to survive harsh environmental conditions in a dormant state.
• Heat is applied because most endospores are resistant to stain.
• Endospores form inside cells and can be free, subterminal, central, central swollen, or terminal swollen.

Capsules

• A coat of uncharged polysaccharides secreted by bacteria, usually round or oval.
• Capsules appear clear after staining.
• Only certain bacterial species have the genes to form capsules.
• Capsule size depends on nutrients in the growth media.
• Capsules should stain pink with a clear halo.
• Capsules increase virulence by preventing phagocytosis by white blood cells.

Motility

• Motile bacteria move using flagella or by gliding.
• Motility can be determined using a hanging drop, flagella stain, or semisolid agar deep.
• Flagella are thin and fragile.
• They can be peritrichous (all around), or polar (at one or both ends).
• Polar flagella can be monotrichous, lophotrichous, or amphitrichous.
• A mordant is used to coat the flagella prior to staining for enhanced visibility.

Staining Errors and Corrections

• Forgetting iodine in a Gram stain causes bacteria to appear pink because crystal violet washes away.
• Crystal violet will wash away during decolorization if iodine is forgotten.
• Negative staining is more accurate than direct staining because it doesn't require heat fixing, which distorts cells.
• Darkfield microscopy gives a similar appearance to negative staining.
• Acidic congo red, like nigrosin, can give a negative stain effect due to its negative charge repelling from bacterial cells.
• Treponema denticola can be seen without distortion using negative staining because no heat is used.

Stain Colors

• Gram-negative cells stain pink or red.
• Gram-positive cells stain purple or blue.
• After an endospore structural stain, endospores appear green, while vegetative cells appear pink or red.
• After a capsule structural stain, cells will be purple with a clear halo against a dark background.
• The capsule itself appears colorless.

Structural Details of Stains

• Capsule structural stain: Congo red
• Endospore structural stain: Safranin

Bacterial Shapes and Structures

• Most flagellated bacteria are rod-shaped bacilli.
• In endospore staining, malachite green is water soluble and easily washes away during decolorization, counterstain (safranin) is retained by the cell bodies.

Pathogenicity

• Capsules contribute to pathogenicity by preventing phagocytosis.
• Flagella contribute to pathogenicity by providing motility, enabling bacteria to move, attach, and colonize.

Endospores and Clostridium

• Endospores enhance Clostridium's survival by resisting harsh conditions and remaining dormant until favorable conditions arise.

Crystal Violet Retention

• In flagella staining, mordant is not used in the stain
• Only Gram-positive cells retain crystal violet because of structural differences.

Endospore Staining of Mycobacterium

• An endospore stain of Mycobacterium would appear green.

Bacterial Capsules

• A culture medium rich in polysaccharides can increase the size of a bacterial capsule.
• Encapsulated Streptococcus stained with safranin and nigrosin will appear as small, red, circular cells in chains with a clear halo against a dark background.

pH

• pH measures hydrogen ion concentrations to determine if fluids are acidic, basic, or neutral.
• A pH scale helps us classify microbes based on their growth in different pH concentrations..
• Each unit of pH difference represents a tenfold change in hydrogen ion concentration.
• Extreme pH levels can affect solubility, denature proteins/enzymes, and disrupt membrane transport and potential.
• Most bacteria prefer a neutral pH (neutrophils).
• Fungi prefer acidic environments.
• Archaea can be found in all pH ranges.
• Microbes maintain a near-neutral internal pH.
• Buffers stabilize pH in growth media.
• Fermentation of sugars can produce acids, altering the pH and preventing other microbes from growing.
• pH changes can classify microbes as alkaliphiles, neutrophiles, or acidophiles, based on their growth preferences.

Microscopy Techniques

• Petroleum jelly prevents evaporation in hanging drop procedures.
• Microorganisms are hard to see in wet preps due to lack of color and blending with water.
• Oil immersion lens cannot be used in hanging drop procedures due to slide thickness.
• Protozoa are easier to see with staining.
• Fine adjustment should be enough to adjust slide when switching between objectives.

Light and Magnification

• More light is needed at higher magnifications, and can be increased using the illuminator or iris diaphragm.
• The illuminator and iris diaphragm control the amount of light reaching the ocular lens.
• Increased magnification reduces the field of vision.
• Parafocal objectives allow easy switching between magnifications.
• The oil immersion objective focuses closest to the slide.

Resolution

• Higher numerical aperture and lower wavelength of light enhance resolving power.
• Low-power objectives are preferred over oil immersion for viewing fungi/algae because they show more of the overall structure.
• Water instead of immersion oil will cause blurry and distorted images due to light bending.

This lesson covers essential microbiology lab techniques. Includes streak plate method for isolated colonies. Also covers inoculation, microscopy, and bacterial culture analysis.