Labs 6-11 Flashcards
Why is it important to prepare thin smears
The thickness of the smear will determine if you can visualize individual cells, their arrangement, or details regarding microstructures associated with cells
Name several goals in preparing a smear
To cause the cells to adhere, to insure that shrinkage of cells does not occur, to prepare thin smears
Why is it important to be sure to cool the loop completely before inserting it into a medium
A loop that is too hot Will spatter the medium and move bacteria into the air
Describe the procedure for making a bacterial smear using a liquid media
To loopfuls of liquid containing organisms are placed on the slide; the smear is allowed to dry at room temperature; slide is passed through flame several times to heat-kill and fix organisms to the slide
What is the first step in the procedure for making a bacterial smear using a solid media
To loopfuls of sterile distilled water are placed on the slide
Name three of the most commonly used dyes for simple staining
Methylene blue, basic fuchsin, and crystal violet
Why do dyes work well on bacteria
Because they have color bearing ions that are positively charged
What is another word for color bearing ions
Chromophores
What three physical characteristics can be determined by simple staining
Morphology, size and arrangement
Negative stains are _____ and thus have a ____ charged chromophore
Negative stains are acidic and thus have a negatively charged chromophore
Two examples of a negative stains
India ink and nigrosin
Negative staining can be useful for studying what and why
Cell dimensions because heat fixation is not performed no shrinkage of cells occurs and size determinations are more accurate
Three examples of when negative staining is more useful
Size determination, capsule observation, observing spirochetes
Which cell has a thicker PG layer
Gram-positive
Which cell has an outer membrane
Gram-negative
What is the primary stain in gram staining and color results
Crystal violet; both cells stain purple
What is the mordant in gram staining and color results
Grams iodine; both cells stain purple
What counterstain is used in gram staining and color results
Safranin; +=purple, -=pink
What is used for decolorization in gram staining and color results
Ethyl alcohol; +=purple, -= “clear”
Final color results of gram staining
Gram-positive will be purple, gram-negative will be pink
What significant difference in structure determines whether the dye-mordant complex is removed from the Gram-negative cell or remains associated with the gram-positive cell
Thickness of the PG layer that comprises the cell wall as well as the presence of an outer membrane
Can gram-negative bacteria ever convert to Gram-positive bacteria
No
Why is it important to prepare thin smears
Thin smears allow the observation of individual cells and any arrangement in which the cells occur; thickness also affects the colorization
What is the most critical step in the Gram stain procedure
Decolorization
What happens if the Destaining reagent is over applied
The dye-mordant complex can eventually be removed from gram-positive cells converting them to gram-negative cells
Explain the gram staining technique
crystal violet for 1 min; rinse with water; gram’s iodine for 1 min; rinse with ethyl alcohol 10-20 secs;rinse with water; safranin for 1 min; rinse with water; blot dry with bibulous paper
Describe the shape and color of B. megaterium
Purple Rods in a long chain (gram-positive)
What 3 genera of medical importance produce endospores
Bacillus, clostridia, sporosarcina
Describe the shape and color of M. catarrhalis
Large pink diplococci
(gram-negative)
If nutrients once again become available the endospore can go through the process of ____
Germination to form a new vegetative cell and growth will resume
Describe endospores
Very dehydrated; not actively metabolizing; resistant to heat, radiation, acids, and many chemicals
Why are endospores resistant
Exosporium (A protein coat that forms a protective barrier around the spore)
The Schaeffer-Fulton spore stain method has four steps describe the first
Cover with small piece of paper towel and saturate with malachite green. Steam for 10 minutes. Add additional stain if stain boils off
What are the functions of calcium dipicolinate and spore specific proteins during endospore formation
Form a cytoplasmic gel that reduces the protoplasmic volume of the endospore to a minimum
The Schaeffer-Fulton spore stain method has four steps describe the 3rd and 4th (last)
Counterstain with safranin for about one minute. Rinse briefly with water to remove safranin. Blot dry with bibulous paper
What does contraction of the thick cortex that forms around the endospore result in
A smaller dehydrated structure
Is calcium dipicolinate present in vegetative cells
No
Explain how to destroy endospores by heating
They must be exposed for 15 to 20 minutes to steam under pressure which generates temps if 121 C
Describe the color results of a properly stained spore-former utilizing the Schaeffer Fulton method
A green endospore contained in a Pink sporangium
The Schaeffer-Fulton spore stain method has four steps describe the 2nd
After the slide has cooled sufficiently remove the paper towel place it in the biohazard bucket and rinse the slide with water for 30 seconds
What color are the spores on the Bacillus Megaterium slide when using the Schaeffer-Fulton spore stain method
Green
The Dorner method for staining produces
A red spore within a colorless sporangium
There are six steps to the Dorner method for staining. What is the 1st
Make a heavy suspension of bacteria by dispersing several loopfuls of bacteria in five drops of sterile water
There are six steps to the Dorner method for staining. What is the 2nd-6th
Add five drops of carbolfuchsin to the bacterial suspension, Heat in a beaker of boiling water for 15 minutes; Mix several loopfuls of bacteria in a drop of nigrosin on the slide and spread across slide. allow to dry and examine.
What to genera test positive when acid-fast staining
Mycobacterium and nocardia
What color are the spores when using the Dorner method for staining
Red
Mycolic acid
It waxy material in the cell wall that significantly affects the staining properties and prevents them from being stained by many of the stains used in mcb
Staphylococcus aureus
Non-acid-fast coccus that can also be part of the normal flora of humans as well as a potential pathogen
The acid-fast stain is an important diagnostic tool the identification of what two diseases
Leprosy and TB
Describe the steps to the acid-fast staining method
Cover with carbolfuchsin; steam over boiling water for 5 min; cool; decolorize with acid alcohol for 15 to 20 seconds; rinse with water; methylene blue for 1 min; rinse with water; blot dry
What is the function of a mordant
A chemical that fixes a dye in or on cells by forming an insoluble compound and thereby promoting retention of that dye
Methylene blue is a positive or negative charged chromophore
Positively charged chromophore; basic stain
Describe the appearance of Staphylococcus aureus when gram staining
Clusters, cocci, purple
(gram-positive)
Describe the appearance of E. coli when Gram staining
Pink, rods, gram-negative
What are the primary stain,counterstain, and mordant of the Schaeffer Fulton method of spore staining
Primary stain: malachite green; counterstain: safranin; mordant: heat
What are the primary stain, counter stain, & mordant using the Doerner method of spore staining
Primary stain: carbolfuchsin; counterstain: nigrosin Mordant: heat
Four diseases detected by spore staining and their genera
Anthrax (bacillus);
botulism, tetanus, gangrene all by clostridium
What is the primary stain, counterstain, decolorizer, mordant of the acid-fast staining method
Primary stain: carbolfuchsin; counterstain: methylene blue; Decolorizer: acid alcohol;
Mordant: heat
Color results of acid-fast positive
Red
Color results of acid-fast negative
Blue
Two genera of acid-fast positive bacteria
Mycobacterium & nocardia
E. coli Gram stain and morphology
Negative rods
Bacillus Megatherium Gram stain and morphology
Positive rod, short chains
Staphylococcus aureus Gram stain and morphology
Positive cocci, irregular clusters
Mycobacterium smegmatis Gram stain and morphology
Positive (purple) rods, may be Y shaped or branched
How many micrometers is each space under 40X power
2.5 microns
How many micrometers is each space under 100X power
1 micron
Which is the only anionic stain
nigrosin
3 genera of spore formers (of medical importance)
Bacillus, Clostridium, and Sporosarcina
