Eprac 1: Bacterial Diagnostic Stains Flashcards
Gram +ve bacteria
Peptidoglycan wall
Plasma membrane
Cytoplasm
Gram -ve bacteria
Outer membrane
Peptidoglycan cell wall
Inner membrane/plasma membrane (membrane protein, phospholipid)
Cytoplasm
Gram stain
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)
Staining reagents
Crystal violet (primary stain)
Iodine (mordant)
Alcohol/acetone (decolouriser)
Carbol fuchsin (counterstain)
Gram stain method
- Make film, air dry then heat fix
- Flood slide with crystal violet for 30 sec -> gently wash with distilled water
- Flood slide with Grams iodine for 30 sec -> gently wash with distilled water
- Decolourise with alcohol for few sec. immediately wash gently with distilled water. Thick smears require longer but do not over-decolourise
- Counterstain with carbol fuchsin counterstain then gently wash with distilled water
Mycobacterium
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
Tuberculosis
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
Diagnosing tuberculosis
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
Kinyoun’s stain
Staining reagents:
Kinyoun’s carbol fuchsin
3% acid alcohol
0.3% aqueous methylene blue (counterstain)
Kinyoun’s stain Method
- Smears should be fixed routinely at 75 degrees for 1hr under UV light
- 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
- Wash with distilled water
- 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
- Wash with distilled water.
- Counterstain with 0.3% aqueous methylene blue counterstain
Result of Kinyoun’s stain
Acid fast bacteria appear red against blue background (methylene blue counterstain)
Non-acid fast bacterium stain blue/purple
Diptheria
An acute contagious disease
Caused by gram +ve bacterium Corynebacterium diphtheriae
Organisms are highly pleomorphic organism with no particular arrangement
Diagnosing Diphtheria
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
Albert’s stain
Staining reagents:
Albert’s stain: Toluidine blue, Malachite green, Glacial acetic acid, Alcohol (95% ethanol), Distilled water
Gram’s iodine
Albert’s stain method
- Make film, air dry and heat fix
- Cover slide with Albert’s stain and allow to act for 5 min
- Wash in water and blot dry
- Cover slide with Gram’s iodine and allow to act for 1 min
- Wash and blot dry
Result of Albert’s stain
Metachromatic granules (volutin) stain bluish black
Rest of cell stains green
Capsule staining
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
-ve capsule staining using india ink (method)
- Place drop of india ink on clean slide and mix in loopful of broth culture or small amt of growth from solid medium
- Carefully place cover slip on drop and press down firmly through several layers of blotting paper until ink film becomes very thin and pale
- Be careful of living organisms which exude from beneath cover-slip and discard blotting paper into disinfectant
Result of India ink method
Capsule appears as clear zone between refractile cell outline and dark background
Positive capsule stain using crystal violet. method:
- Place small drop of india ink on one end of clean slide
- Add a loopful of broth culture or small amt of growth from solid medium
- Use second slide to drag mixture across slide, creating thin film
- Allow smear to air dry then stain with 1% crystal violet for 1-2 mins
- Drain crystal violet off, tilting slide at 45 degree angle. Allow to air dry
Result of crystal violet capsule stain
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
Bacterial spores
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)
Scaeffer/fulton spore stain
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
Scaeffer/fulton spore stain: Method
- Flood side with Malachite green and steam for 1 min
- Wash under running water
- Counterstain with Safranin for 30-60 secs
- 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)
