Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MICR2208.2 > Eprac 1: Bacterial Diagnostic Stains > Flashcards

Eprac 1: Bacterial Diagnostic Stains Flashcards

1
Q

Gram +ve bacteria

A

Peptidoglycan wall

Plasma membrane

Cytoplasm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Gram -ve bacteria

A

Outer membrane

Peptidoglycan cell wall

Inner membrane/plasma membrane (membrane protein, phospholipid)

Cytoplasm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Gram stain

A

Before staining (all colourless)

Crystal violet -> all cells purple

Gram’s iodine -> all cells dark purple (iodine added as a mordant (fixative) . Forms insoluble complex with crystal violet)

Alcohol -> gram +ve cells: purple; gram -ve cells: colourless (Decolourisation step: differentiating strep. Gram +ve: thick peptidoglycan cel walls which retain crystal violet/iodine dye. Gram -ve have thin peptidoglycan cell walls surrounded by layer of lipopolysaccharide. Cells leach dye when exposed to alcohol)

Carbol fuschin -> gram +ve cells purple; gram -ve pink (counterstain step-> give colour to cells rendered by colourless by alcohol -> make them visible under light microscopy)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Staining reagents

A

Crystal violet (primary stain)

Iodine (mordant)

Alcohol/acetone (decolouriser)

Carbol fuchsin (counterstain)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Gram stain method

A
  1. Make film, air dry then heat fix
  2. Flood slide with crystal violet for 30 sec -> gently wash with distilled water
  3. Flood slide with Grams iodine for 30 sec -> gently wash with distilled water
  4. Decolourise with alcohol for few sec. immediately wash gently with distilled water. Thick smears require longer but do not over-decolourise
  5. Counterstain with carbol fuchsin counterstain then gently wash with distilled water
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Mycobacterium

A

Comprises acid-fast bacilli -> difficult to stain (once stained resist decolourisation with acid alcohol)

Waxy mycolic acid in cell envelop: resistant to drying which improves survival in environments. Binds carbol fuschin and cannot be removed with acid-alcohol during decolourisation step

Called acid fast bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Tuberculosis

A

Caused by mycobacterium tuberculosis, acid fast bacterium

Highly infectious by inhalation

Droplet nuclei (coughed up from infected lungs) may float in air for several hours. 1-5um particles (consisting of 1-10 bacteria) are of such size that they evade innate defences of respiratory tract and are inhaled to level of alveoli

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Diagnosing tuberculosis

A

Demonstrate presence of tubercle bacilli in clinical specimen, most commonly from sputum

Sputum smear examined for presence of acid-fast bacteria, by Kinyoun’s stain

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Kinyoun’s stain

A

Staining reagents:

Kinyoun’s carbol fuchsin

3% acid alcohol

0.3% aqueous methylene blue (counterstain)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Kinyoun’s stain Method

A
  1. Smears should be fixed routinely at 75 degrees for 1hr under UV light
  2. Flood slide with Kinyoun’s carbol fuchsin. Stain for 10 min without heating. Do not allow slide to dry out but flood again if necessary. Do not reheat slide
  3. Wash with distilled water
  4. Decolourise with 3% acid alcohol until no more colour is seen in washings (about 2 mins). Thick smears may require longer but do not over-colourise
  5. Wash with distilled water.
  6. Counterstain with 0.3% aqueous methylene blue counterstain
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Result of Kinyoun’s stain

A

Acid fast bacteria appear red against blue background (methylene blue counterstain)

Non-acid fast bacterium stain blue/purple

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Diptheria

A

An acute contagious disease

Caused by gram +ve bacterium Corynebacterium diphtheriae

Organisms are highly pleomorphic organism with no particular arrangement

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Diagnosing Diphtheria

A

Albert’s stain used to demonstrate metachromatic granules; formed in polar regions

Differential plate known as tinsdale agar is used to identify C.diptheriae

C.diptheriae colonies produce black halo around colony

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Albert’s stain

A

Staining reagents:

Albert’s stain: Toluidine blue, Malachite green, Glacial acetic acid, Alcohol (95% ethanol), Distilled water

Gram’s iodine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Albert’s stain method

A
  1. Make film, air dry and heat fix
  2. Cover slide with Albert’s stain and allow to act for 5 min
  3. Wash in water and blot dry
  4. Cover slide with Gram’s iodine and allow to act for 1 min
  5. Wash and blot dry
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Result of Albert’s stain

A

Metachromatic granules (volutin) stain bluish black

Rest of cell stains green

17
Q

Capsule staining

A

To distinguish capsular material from bacterial cell, a capsule stain may be perform

Various methods for visualising capsules by direct microscopic examination

Presence of capsules: best observed in actively growing broth culture. Specific broth media and conditions may be required for capsules to be clearly visible when stained

18
Q

-ve capsule staining using india ink (method)

A
  1. Place drop of india ink on clean slide and mix in loopful of broth culture or small amt of growth from solid medium
  2. Carefully place cover slip on drop and press down firmly through several layers of blotting paper until ink film becomes very thin and pale
  3. Be careful of living organisms which exude from beneath cover-slip and discard blotting paper into disinfectant
19
Q

Result of India ink method

A

Capsule appears as clear zone between refractile cell outline and dark background

20
Q

Positive capsule stain using crystal violet. method:

A
  1. Place small drop of india ink on one end of clean slide
  2. Add a loopful of broth culture or small amt of growth from solid medium
  3. Use second slide to drag mixture across slide, creating thin film
  4. Allow smear to air dry then stain with 1% crystal violet for 1-2 mins
  5. Drain crystal violet off, tilting slide at 45 degree angle. Allow to air dry
21
Q

Result of crystal violet capsule stain

A

Cells are stained deep blue or purple

Capsules stain light blue against dark background

Even narrow capsules are visible since shrinkage associated fixation is avoided

22
Q

Bacterial spores

A

Sporulation: in poor growth conditions, some bacteria (Bacillus and clostridium) produce resistant survival forms: endospores -> vegetative portion of bacterium is degraded and dormant endospore is released -> germinates, producing single vegetative bacterium

Do not serve as reproductive function

Resistant to extreme environmental conditions (high temp, dryness, toxic chemicals and UV radiation) -> able to survive long period of time

Can be killed by sterilisation methods (autoclaves, hot air ovens)

23
Q

Scaeffer/fulton spore stain

A

More vigorous stain

If gram stain used on spore-bearing organism -> spore appears colourless (resistant to staining)

Staining reagents: 5% aqueous malachite green, 0.5% aqueous Safranin

24
Q

Scaeffer/fulton spore stain: Method

A
  1. Flood side with Malachite green and steam for 1 min
  2. Wash under running water
  3. Counterstain with Safranin for 30-60 secs
  4. Rinse with water and blot dry

Can be used as cold stain by allowing malachite green to act for 10 min at room temp

-> bacterial bodies: red; spores: green

Once spores are stained: can be described based on position within bacterium and whether they cause distension (stretching of cell)

MICR2208.2 (16 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions