Mirco Lab Topic 3

Describe a biological stain (give the three components and what they do)

1. Benzene- organic colorless solvent

2. Chromophore - chemical group that imparts color to the benzene

3. Auxochrome - chemical group that conveys the property of ionization to the chromogen, enabling it to form salts and bind to fibers or tissues

Define a simple stain

1. uses a single dye

2. has a positive charge

3. allows for observation of the morphology

define a differential stain (aka acidic)

1. uses more than one dye

2. has a negative charge

3. allows observation of morphology and differences in cell structure

give examples of positive stains

1. Methylene blue

2. crystal violet

3. safranin

4. malachite green

How does a positive stain work? (aka basic)

• cytoplasm attracts and absorbs the dye, and appears stained

• stain bacterial cells directly

• all cells appear the same color

Describe an gram satin

1. Type of differential stain

2. allows for differentiation of Gram + and Gram - cells based on peptidoglycan concentration

Describe an acid fast stain

1. A type of differential stain

2. detects the presence of mycolic acid layer in the cell wall

describe an endospore stain

1. A type of differential stain

2. differentiates between vegetative cells and endospores

why is a negative stain done? **

1. To observe cells in their natural state, as positive staining can damage them

2. to observe cell structures that are impermeable to a positive stain such as endospores and capsules

Describe the cocci morphology

• round cells

• arrangements: diplococci (paris), streptococci (chains), staphylococci (clusters)

describe the bacillus morphology

• rod-shaped cells

• arrangements: diplobacilli, coccobacilli…

describe the spirilla morphology

• spiral or curved rods

• spirillum, spirochetes, vibrio

List the steps for how to make a smear on a glass slide from slant cultures

1. Flame the inoculating loop to avoid cross-contamination

2. Using the loop, place a drop of sterile water on a clean glass slide

3. Flame the inoculating loop

4. Allow the loop to cool

5. Using the loop, transfer a pinpoint amount of bacteria onto the slide

6. Mix to create a thin and uniform smear

7. allow the semar to air dry

What is the purpose of a clean glass slide

reduces cross contamination

what is the purpose of the thin uniform smear

a thin uniform smear allows light to pass through and ensures the stain will dry evenly

what is the purpose of air drying a smear

1. air drying prevents the distortion of cells

2. if the smear is still wet, the water may boil upon heat fixing, and rupture the cells

what is the purpose of heat fixing a slide

1. adheres the bacteria to the slide

describe the morphology and arragenment for Staphylococcus epidermidis

spherical shaped cells called cocci arranged in clusters that are referred to as staphylo

describe the morphology and arrangement for Escherichia coli

1. rod-shaped

2. appears in short chains

Discuss the implications of capsules in bacterial diseases

1. Capsules allow for attachment

2. capsules help bacteria from biofilm which can lead to persistent infections like dental plaque

can a negative stain show the internal structures of a bacterial cell

no, they do not penetrate the cell and only show structures like cell shape, size and capsules

Is coffee a biological stain or just a compound

1. coffee is not a biological stain because it does not contain auxochrome

Name four positive stains

1. methelyene blue

2. crystal violet

3. safranin

4. Malachite Green

Name two negative stains

1. nigrosin

2. congo red

What is a mordent

1. A mordant is a chemical used in staining that helps the primary stain bind to the specimen

Decolorizing

a decolorizing agent is substance used in staining that removes excess primary stain from cells, which helps differentiate between different types of microorganisms

What is a counterstain

This is a secondary dye applied after decolorization to color cells that did not retain the primary stain, making them easier to distinguish under a microscope

Describe the differences between gram - and gram +

• Gram-positive cells have thick peptidoglycan in the cell wall

• Gram-negative thin peptidoglycan and outer membrane

List the Gram staining steps in order

1. Place the slide with the smear side up on the staining rack

2. Cover the smear with crystal violet and let it sit for 1-2 minutes

3. Wash the slide with distilled water, and remove the stain

4. Cover the smear with Gram’s iodine and let it sit for 1-2 minutes

5. Rinse again with distilled water and drain off excess water

6. decolorize by washing the slide briefly with ethyl alcohol for 2-3 seconds

7. Immediately end decolorizing by rinsing with distilled water

8. Cover the smear with safranin counterstain for 1-2 minutes

9. rinse slide with water

10. blot dry with bibulous paper and allow slide to dry thoroughly

11. examine slide with immersion oil under microscope

what is the most important step in the gram staining procedure

The decolorizing step is the most crucial because it determines whether the bacteria will remain purple (gram+) or lose the primary stain and become pink after the counterstain

what would be the appearance of the bacteria if you forgot the counterstain with safranin

gram negative bacteria would appear colorless while gram positive bacteria would remain purple

what would be the appearance of the bacteria if you used methylene blue instead of safranin

gram negative bacteria would stain blue instead of pink while gram positive would remain purple

what would be the appearance of the bacteria if the mordant was not used

Gram-positive bacteria would loose the crystal violet stain, and they may appear red, making them look falsely like gram negative cells because they will take up the safranin instead

what would be the appearance of the bacteria if the decolorizing step was too long, too short or omitted

too long- Gram-positive bacteria would become pink due to over-decolorization

too short- gram-negative bacteria may stay purple

omitted- both gram-negative and gram-positive bacteria would appear purple

what is the principle of differential staining

different types of cells or microorganisms stain differently based on differences in their cell structure, which allows them to be distinguished under a microscope

the gram stain helps observe which specific bacterial cell anatonmy

their cell wall

gram positive cells have thick peptidoglycan in their cell walls and gram negative cells have thin peptidoglycan

what is gram variability

This occurs when bacteria do not stain as expected, and they do not appear exaclty purple or pink

Describe 3 conditions that may result in a Gram positive organism staining gram negative

1. age of the culture: cultures past the ideal time of 18-24 hrs have weakened cell walls, which cause gram-positive cells to lose the crystal violet and appear gram-negative

2. Over decolorization: Leaving the decolorizer on too long can strip the primary stain from Gram-positive cells

3. damage to the cell wall: physical, chemical or environmental damage can make Gram-positive bacteria unable to retain the primary stain

what is the medical importance for a gram stain

1. This is the first step in diagnosis

2. this is crucial in the prescription of the right antibiotics

list 2 gram positive bacteria and their disease

1. Staphylococcus aureus- skin infections

2. staphylococcus pyogenes- strep throat

list 2 gram negative bacteria and their diseases

1. Escherichia coli- UTI

2. Salmonella- food poisoning

Which is more difficult to treat- infection caused by Gram negative or Gram positive cells

Gram-negative infections are more difficult to treat because they have an outer membrane that acts as a barrier to many antibiotics

On a Friday evening, the health care personnel on duty forgot to refrigerate the urine sample and left it on the counter. On Monday, the sample was sent to the lab and the Gram stain showed great variability ranging from intense purple to shades of red. How would you account for this result?

1. Leaving the urine unrefrigerated will allow bacterial overgrowth, death, and cell wall degradation

2. Gram-positive cells will lose the cyrstal violet stain

3. This will result in gram variability

What is the primary stain for an acid fast cell

1. carbol fuschin

2. this colors acid fast bacteria red by penetrating their waxy cell walls with mycolic acid

what is the decolorizing agent in an acid fast stain

1. acid alcohol

2. removes the primary stain from non-acid-fast cells

What is the secondary/counterstain for acid fast cells

1. Methylene blue

2. colors non acid fast cells blue to be distinguished from red acid fast bacteria

What is the difference in the cell wall of acid fast cells vs non

1. Acid-fast cells have a thick, waxy cell wall that contains mycolic acid, this makes them resistant to decolorization and able to retain a primary stain

2. non acid fast cells have normal peptidoglycan so they loose the primary stain and take up the counterstain

why is heat or phenol used with application of primary stain during acid fast staining

1. heat or phenol is used in order to help penetrate the thick mycolic acid in the cell wall of the acid fast bacteria so that carbol fuchsin can penetrate and stain

What is used for decolorizing the primary stain?

1. acid alcohol is used because the strong acid helps remove the carbol fuchsin from the non acid past cells while allowing acid fast cells to retain their stain due to their waxy cell walls

Which specific bacterial anatomy is observed with the acid-fast stain?

Mycolic acid rich cell wall

What is the diagnostic value of the acid-fast stain?

helps differentiate acid fast bacteria like Mycobacterium species, which are responsible for diseases like tuberculosis or Hansens disease

Identify two acid-fast bacteria and the diseases they cause

1. Mycobacterium tuberculosis = Tuberculosis

2. Mycobacterium leprae = Leprosy

Identify the unique molecule found in the cell wall of an acid-fast bacteria and explain and explain its significance in the resistance of Mycobacteria sps.

1. Mycolic acid in the cell wall is unique

2. This makes the cell wall thick and impermeable, which leads to resistance to many drugs or antimicrobial methods