Comprehensive Bacterial Staining, Identification, and Culture Techniques

What is the objective of simple staining in microbiology?

To understand the application of positive and negative stains and to show a difference between the organism and background.

What are the basic dyes used in positive staining?

Methylene blue, basic fuchsin, and crystal violet.

How do positive chromophores interact with bacterial cell walls during positive staining?

Positive chromophores are attracted to the negatively charged bacterial cell walls, resulting in the bacteria being stained.

What is the purpose of negative staining?

To visualize cell morphology by using negatively charged chromophores that are repelled by the negatively charged cell wall.

What are the acidic dyes used in negative staining?

Eosin and Nigrosin.

Why does negative staining not require heat fixing?

Because heat can distort the bacterial cell shape and size.

What is the procedure for simple staining?

Add bacterial suspension to a slide, air dry, heat fix, add methylene blue, let sit for 1-2 minutes, and rinse off excess stain.

What are the results of staining E. coli, B. subtilis, S. epidermidis, and B. megaterium?

E. coli: Spaced out purple rods; B. subtilis: Purple rods, some spaced out, some in clusters; S. epidermidis: Purple cocci, spaced out and in clusters; B. megaterium: White swirls.

What are common errors in the simple staining procedure?

Not air drying completely before heat fixing, over/under heat fixing, and excessive washing.

What is the objective of gram staining?

To learn the difference between gram positive and gram negative bacteria based on cell wall structure.

What characterizes gram positive bacteria?

They have a thick peptidoglycan layer with no outer membrane, which traps the stain and keeps them purple after staining.

What characterizes gram negative bacteria?

They have a thin peptidoglycan layer with an outer membrane rich in lipopolysaccharides, causing the stain to wash out during decolorization.

What is the role of iodine in the gram staining procedure?

Iodine acts as a mordant, forming a crystal violet-iodine (CV-I) complex that makes the stain less soluble.

What happens to gram positive cells during the decolorization step?

Alcohol dehydrates and shrinks the thick peptidoglycan, trapping the crystal violet-iodine complex, so they remain purple.

What happens to gram negative cells during the decolorization step?

Alcohol dissolves the outer membrane lipids and washes out the CV-I complex, making the cells colorless.

What is the final step in the gram staining procedure?

Adding safranin for 1 minute to provide visibility, staining gram negative cells red/pink.

What are the results of staining E. coli in the gram staining procedure?

E. coli appears rod-shaped and stained red/pink, indicating it is gram negative.

What is the significance of chromophores in staining?

Chromophores are charged molecules responsible for the stain's color, influencing how they interact with bacterial cell walls.

What are the shapes of bacteria observed in Experiment 3?

Rods (bacilli), spheres (cocci), and spirals.

What is the importance of air drying in the staining process?

Air drying prevents distortion of the bacterial cells before heat fixing.

What is the purpose of heat fixing in staining?

Heat fixing adheres the bacteria to the slide and kills the cells, preserving their structure for observation.

What is the role of safranin in the gram staining procedure?

Safranin serves as a counterstain, providing visibility to gram negative cells after decolorization.

What shape are Staphylococcus epidermidis cells and how do they stain?

Cocci-shaped cells that stain purple, indicating they are Gram positive.

What happens to older Gram positive cells during staining?

They lose cell wall integrity, causing the peptidoglycan layer to break down, which can lead to a false Gram negative result.

What is the role of the mordant in Gram staining?

The mordant binds to the crystal violet to form a large CV-I complex that gets trapped in the thick cell wall.

What occurs if there is no decolorizer used in the Gram staining process?

All cells, regardless of being Gram positive or negative, will appear purple.

What is the effect of over-decolorization in Gram staining?

It removes too much stain, making the cells appear pink/red, indicating they are Gram negative.

What is the objective of the Kinyoun method in microbiology?

To differentiate between acid-fast and non-acid-fast bacteria.

What is unique about the cell wall of Mycobacterium species?

It contains a waxy layer rich in mycolic acid, which resists simple and Gram staining.

How does the Kinyoun method allow for stain penetration?

It uses high concentrations of phenol and carbolfuchsin to penetrate the waxy cell wall without heat.

What is the primary stain used in the Kinyoun method?

Carbolfuchsin, which stains acid-fast cells red.

What is the decolorizing agent in the Kinyoun method?

Acid alcohol, which removes dye from non-acid-fast cells.

What counterstain is used in the Kinyoun method and what does it stain?

Methylene blue, which stains decolorized non-acid-fast cells blue.

What are the results for acid-fast organisms using the Kinyoun method?

They appear pink/red and are typically clustered rods.

What are the results for non-acid-fast organisms using the Kinyoun method?

They appear blue and are typically cocci in grape-like clusters.

What is the purpose of spores in bacteria?

Spores are survival structures formed under unfavorable conditions, not for reproduction.

What is sporogenesis?

The process involving DNA replication, cell division, engulfment of forespores, formation of a thick protein coat, and the death of the vegetative cell.

What makes spores resistant to various environmental factors?

Their keratin coat makes them impermeable to most dyes, heat, chemicals, and radiation.

What is the primary stain used in spore staining?

Malachite green.

What is the decolorizer used in spore staining?

Water.

What counterstain is used in spore staining?

Gram's safranin.

What is the difference between the cold and hot methods of spore staining?

The cold method requires no steaming and uses high dye concentration, while the hot method uses steam to push in malachite green.

Which genera commonly form endospores?

Bacillus and Clostridium.

What is the objective of negative staining?

To visualize bacteria using an acidic dye.

What is a key step in the procedure for negative staining?

The slide is air-dried and not heat-fixed.

What is the objective of Experiment 8?

To differentiate bacteria with capsules from those without one by using a combination of positive and negative staining techniques.

What are capsules in bacteria composed of?

Polysaccharide-protein complexes surrounding some bacteria.

Why can't capsules be stained with simple stains?

Capsules are non-ionic and water soluble, so they do not take up simple stains.

What two stains are used in Experiment 8?

Nigrosin and crystal violet.

What does nigrosin stain in the negative staining technique?

The background.

What does crystal violet stain in the negative staining technique?

The bacterial cell wall but not the capsule.

What is the appearance of the capsule when using negative staining?

A clear halo around the cell, as it remains unstained due to its non-ionic nature.

What is the purpose of selective media in bacterial culture?

To contain chemicals that prevent the growth of unwanted bacteria, allowing the growth of target bacteria.

What is the difference between selective and differential media?

Selective media prevents the growth of unwanted bacteria, while differential media contains dyes or indicators to distinguish bacteria based on metabolic or biochemical properties.

What is enriched media used for?

To contain nutrients like blood or serum to support fastidious organisms.

What is an example of a general-purpose medium?

Tryptic Soy Agar (TSA), which supports many bacteria.

What does Eosin Methylene Blue (EMB) agar do?

It is selective (inhibits Gram-positive bacteria) and differential (lactose fermentation indicated by colony color).

What type of medium is KF Streptococcal Agar?

It is both selective and differential, targeting streptococci.

What is the first step in the procedure for using agar plates in Experiment 10?

Divide the agar plate into quadrants and smear.

How long should the agar plates be incubated in Experiment 10?

For 48 hours.

What is the role of methylene blue in staining?

It cannot be used in place of nigrosin because it is a positively charged (cationic dye).

What are the advantages of negative staining?

Cell morphology is not distorted.

Why are capsules hard to stain?

Because they are non-ionic and water soluble, preventing dye from binding to them.

What color does the bacterial cell wall appear after staining with crystal violet?

Purple.

What color does the background appear after using nigrosin?

Dark.

What is the significance of a clear halo around the bacterial cell in negative staining?

It indicates the presence of a capsule that does not take up the stain.

What is the purpose of the Streak Plate Method (Experiment 11)?

To isolate pure bacterial colonies by spreading bacteria on agar surface.

What is the principle behind the Streak Plate Method?

Surface dilution by physically spreading bacteria to separate cells.

Where does colony growth occur in the Streak Plate Method?

Colonies grow only on the agar surface.

What is the ideal result of the Streak Plate Method?

Well isolated colonies in later streak zones.

What is the purpose of the Pour Plate Method (Experiment 12)?

To isolate pure colonies by mixing bacteria into molten agar and solidifying.

What is the principle behind the Pour Plate Method?

Serial dilution in molten agar to separate individual cells throughout the medium.

Where does colony growth occur in the Pour Plate Method?

Colonies grow within and on the agar.

What is the ideal result of the Pour Plate Method?

Single isolated colonies distributed throughout the agar.

What is the purpose of sterilization in microbiology (Experiment 13)?

To prevent contamination of cultures, transmission of pathogens, maintain integrity of experiments, and prevent spoilage.

What does sterile/sterilized mean in microbiology?

Completely free of all life, including viruses.

What are common methods of sterilization?

Physical methods (heat, radiation, filtration) and chemical methods (disinfectants, antiseptics).

What is the main tool for sterilization in microbiology?

Autoclave.

What are the conditions for steam sterilization in an autoclave?

121°C for 15-20 minutes.

What indicates a sterile broth after incubation?

Clear, no turbidity, no sediment, no gas.

What indicates a non-sterile broth after incubation?

Cloudy/turbid, possible sediment, gas bubbles, pellicle, or surface growth.

What is the objective of the Standard Plate Count (SPC) method (Experiment 15)?

To estimate the number of viable bacteria in a sample.

How does the SPC method work?

Repeatedly dilute a sample, plate a small amount on agar, incubate, and count the number of colonies formed.

What is the output of the SPC method?

Number of colony-forming units per mL, estimating the number of viable bacteria in the original sample.

What is the difference between SPC and Direct Count Method?

SPC counts only viable bacteria that grow into colonies, while Direct Count counts total bacteria (alive + dead) using a microscope.

What is an advantage of the SPC method?

Only counts live bacteria that form colonies.

What is a disadvantage of the SPC method?

Bacteria can only grow under certain conditions, and counting can be difficult if colonies overlap.

If 0.1mL of a 1 x 10^-6 dilution plate contains 56 colonies, how do you calculate the number of cells per mL of the original culture?

Multiply the number of colonies by the dilution factor (56 colonies x 10^6 x 10).

What is the concentration of colonies if there are 56 colonies in 0.1 mL?

560 colonies.

What is the calculation to find the number of colonies if dilution is not given?

56 colonies / 0.1 mL x 10^-6 = 5.6 x 10^8.

Which dilution had the most concentrated number of colonies?

10^-4.

Which dilution barely had any colonies?

10^-9.

What is the objective of Experiment 16: Demonstration of Motility?

To differentiate motile and non-motile bacteria by observing their movement.

How do motile bacteria move?

They rotate their flagella to move actively.

What causes non-motile bacteria to appear to move?

Brownian motion (random vibrations caused by water molecule collisions).

Describe the appearance of movement in motile bacteria.

Smooth, purposeful zigzag or directional movement across the medium.

Describe the appearance of movement in non-motile bacteria.

Vibrating or shaking in place without true movement.

What is the purpose of motility test agar?

It allows motile bacteria to move through the medium due to its semi-solid nature.

What happens to motile bacteria in motility test agar after incubation?

They diffuse away from the stab line, making the medium turbid beyond the stab.

What is the procedure for the motility test agar?

Stab the bacteria straight down the center with an inoculation needle without wiggling.