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 differentiate between the organism and background.

What is the purpose of using positive chromophores in staining bacteria?

Positive chromophores cause positive staining since the negatively charged bacterial cell wall takes up the dye, resulting in a blue stain.

Name three basic dyes used for positive staining.

Methylene blue, basic fuchsin, and crystal violet.

What is the effect of using negatively charged chromophores in staining?

They are repelled by the negatively charged cell wall, leading to negative staining which helps visualize cell morphology.

List two acidic dyes used for 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 in simple staining?

Spaced out purple rods.

What common errors can occur during 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 distinguishes gram positive bacteria from gram negative bacteria?

Gram positive bacteria have a thick peptidoglycan layer with no outer membrane, while gram negative bacteria have a thin peptidoglycan layer with an outer membrane rich in lipopolysaccharides.

What is the primary stain used in gram staining?

Crystal violet.

What role does iodine play 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 layer, 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 counterstain used in gram staining?

Safranin.

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

Rod shaped, stained red/pink, indicating it is gram negative.

What is the first step in the gram staining procedure?

Prepare a bacterial smear on a clean slide, air dry, and heat fix.

How long should the crystal violet be applied during gram staining?

For 1 minute.

What is the effect of the mordant in the gram staining process?

It forms a complex that makes the crystal violet less soluble, helping to retain the stain in gram positive cells.

What is the purpose of rinsing with water after each staining step in gram staining?

To remove excess stain and prevent interference in the results.

What characteristic shapes of bacteria are observed in Experiment 3?

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

What shape are the cells of Staphylococcus epidermidis 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 peptidoglycan layer.

What occurs if no decolorizer is used during Gram staining?

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

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

It removes too much stain, making Gram positive cells appear pink/red, similar to Gram negative cells.

What is the objective of the Kinyoun method?

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

What is unique about the cell wall of Mycobacterium species?

It contains mycolic acid, which is lipid-rich and resists simple and Gram staining.

How does the Kinyoun method differ from the Ziehl-Neelsen method?

The Kinyoun method is a cold stain that uses high concentrations of phenol and carbolfuchsin, while the Ziehl-Neelsen method uses heat as a mordant.

What is the primary stain used in the Kinyoun method?

Carbolfuchsin, which stains 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?

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

What color do acid-fast organisms appear after staining with the Kinyoun method?

Pink/red, as they retain the carbolfuchsin stain.

What color do non-acid-fast organisms appear after staining with the Kinyoun method?

Blue, as they take up the methylene blue counterstain.

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, and formation of a thick protein coat, leading to the death of the vegetative cell.

What makes spores resistant to staining?

The keratin coat makes spores impermeable to most dyes.

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 color do endospores appear after spore staining?

Green, as they retain malachite green.

What color do vegetative cells appear after spore staining?

Red, as they take up the safranin counterstain.

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

The cold method requires no steaming and relies on high dye concentration, while the hot method uses steam to facilitate dye penetration.

What is the objective of negative staining?

To visualize bacteria using an acidic dye without heat fixing.

What is the purpose of using nigrosin in negative staining?

Nigrosin stains the background, allowing for clear visualization of bacterial morphology without distortion.

Why can't methylene blue be used in place of nigrosin?

Methylene blue is a positively charged (cationic) dye, which does not work for negative staining.

What is the objective of Experiment 8?

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

What are capsules in bacteria composed of?

Capsules are polysaccharide-protein complexes surrounding some bacteria.

Why do capsules not take up simple stains?

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

What does crystal violet stain in the staining procedure?

Crystal violet stains the bacterial cell wall but not the capsule.

What are the results of the staining procedure in terms of color?

The background is dark (stained by nigrosin), the bacterial cell wall is purple (stained by crystal violet), and the capsule appears as a clear halo around the cell.

What is the objective of Experiment 10?

To understand how to use selective, differential, and enriched culture media in bacterial growth and identification.

What is chemically defined media?

Chemically defined media has a known chemical composition.

What is complex media?

Complex media contains extracts (like blood or serum) with an unknown exact chemical makeup.

What is selective media?

Selective media contains chemicals that prevent the growth of unwanted bacteria, allowing the growth of target bacteria.

What is differential media?

Differential media contains dyes or indicators to distinguish bacteria based on metabolic or biochemical properties.

What is enriched media?

Enriched media contains nutrients like blood or serum to support fastidious organisms.

What is Tryptic Soy Agar (TSA)?

TSA is a general-purpose medium that supports the growth of many bacteria.

What is Eosin Methylene Blue (EMB) agar used for?

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

What is KF Streptococcal Agar?

KF Streptococcal Agar is a selective and differential medium targeting streptococci.

What is the procedure for using agar plates in bacterial growth experiments?

Divide the agar plate into quadrants, smear the sample, and incubate for 48 hours.

What is the purpose of the Streak Plate Method?

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

What is the purpose of the Pour Plate Method?

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

What is the principle behind the Streak Plate Method?

Surface dilution by physically spreading bacteria to separate cells.

What is the principle behind the Pour Plate Method?

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

Where do colonies grow in the Streak Plate Method?

Colonies grow only on the agar surface.

Where do colonies grow in the Pour Plate Method?

Colonies grow within and on the agar.

What is a common use of the Streak Plate Method?

Routine isolation of pure cultures for identifications.

What aseptic technique is used in the Streak Plate Method?

Flame loop between streaks to dilute bacteria.

What is the ideal result of the Streak Plate Method?

Well isolated colonies in later streak zones.

What does a clear broth indicate after sterilization?

Success (no contamination).

What does a cloudy/turbid broth indicate after sterilization?

Contamination occurred.

What is the main tool used for sterilization in microbiology?

Autoclave.

What is the sterilization process in an autoclave?

Steam sterilization at 121°C for 15-20 minutes.

What are common methods of sterilization?

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

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

To estimate the number of viable bacteria in a sample.

How does the Standard Plate Count method work?

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

What is the output of the Standard Plate Count method?

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

What is the difference between the 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 Standard Plate Count method?

It only counts live bacteria that form colonies.

What is a disadvantage of the Standard Plate Count method?

Bacteria can only grow under certain conditions and can be hard to count if too few or too many.

How would you calculate the number of cells per mL from a dilution?

If 0.1mL of a 1 x 10^-6 dilution plate contains 56 colonies, multiply 56 by the dilution factor (10^7) to find the original concentration.

What is the calculation for determining the number of colonies if 56 colonies are found in 0.1mL?

56 colonies/0.1mL = 560 colonies.

What is the result of the dilution calculation if the dilution factor is not given?

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

Which dilution had the most concentrated number of colonies?

10^-4 had the most amount/concentrated number of colonies.

What was observed regarding colonies at a dilution of 10^-9?

10^-9 barely had any colonies.

How did the colonies appear visually?

Colonies looked like yellow circles.

What is the objective of the motility experiment?

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

How do motile bacteria move?

Motile bacteria rotate their flagella to move actively.

What causes non-motile bacteria to appear to move?

Non-motile bacteria appear to move due to Brownian motion, which is caused by random vibrations from water molecule collisions.

Describe the appearance of movement for motile bacteria.

They exhibit smooth, purposeful zigzag or directional movement across the medium.

What is the appearance of non-motile bacteria?

They vibrate or shake in place without true movement.

What is the purpose of the motility test agar?

It is a semi-solid agar that allows motile bacteria to move through the medium.

What is the procedure for using motility test agar?

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

What indicates that motile bacteria have moved in the motility test agar?

Motile bacteria will diffuse away from the stab line, making the medium turbid beyond the stab.

What happens with non-motile bacteria in the motility test agar?

Non-motile bacteria only grow along the stab line.

What is the hanging drop method used for?

It allows observation of live, unstained bacteria to see motility and arrangement under minimal light.

Why is staining not used in the hanging drop method?

Stains kill bacteria and prevent movement.

What were the observations for Lactobacillus plantarum in the motility tests?

Growth only along the stab line and bacteria vibrate/shake in place, indicating it is non-motile.