MCROBIO 233 CUNY Enterotube Acid Stain and Gram Positive vs Negative Lab Report short lab report on each top on different docs please keep it simple and th

MCROBIO 233 CUNY Enterotube Acid Stain and Gram Positive vs Negative Lab Report short lab report on each top on different docs please keep it simple and thorough SIMPLE STAINING
In order to stain the bacterial specimen for microscopy one must first prepare the smear on
the slide. This basically involves 3 steps—-transferring a liquid suspension of the bacterium
on the slide, drying the smear, and then heating slightly to firmly attach the smear to the slide.
Once this is done, the staining procedure begins.
CAUTION: Do NOT make your smear suspensions too thick: The dye will not penetrate
well, and there will be far too many bacterial cells to see individual shapes and arrangements.
One needs to be careful about thick smears when taking the specimen from an agar medium.
RECOMMENDATION: You may find it helpful to draw a circle (wax pencil is best) on the
opposite side of the slide where you will spread your smear. This will help you later in
locating the smear, sometimes a problem when moving the slide back and forth looking for
your bacteria. The wax pencil is better than a marker because it will not wash off easily from
the glass.
OBJECTIVES:
Prepare a smear from a bacterial specimen.
Prepare a simple stain of the smear.
Use the microscope to identify features (shape, arrangement, size) of the bacterium.
MATERIALS NEEDED:
NB culture of Staphylococcus epidermidis
NA plate of E. coli
dye tub. dye reagents
clean microscope slides
wax pencil
bibulous blotting paper
immersion oil
THE PROCEDURES:
1.
First, make sure that you have a few clean slides. We recycle our microscope
slides and the smears have to be removed from the glass. You will use a cleansing
powder liquid that works well to remove the smear and the dye from the glass.
o At the sink, pick up the bottle of the slide cleansing liquid , pouring some of the
thick suspension onto the slide.
o Use your finger to spread the suspension over each side of the slide, and then
wash WELL with tap water.
o Dry the slide.
2.
You will make smears using 2 different types of cultures, a broth and an agar culture.
o Label the 2 slides with the names of your bacteria.
Shaking the culture first, aseptically transfer a drop of the broth culture using an
inoculating loop to a clean slide.
o From the agar plate culture, remove a small inoculum (use only a small amount
of large colonies) with a loop and suspend it in a small drop of distilled water.
Mix the culture well into the water drop.
3. Label 2 slides—Staph and E. coli.
4. Spread the smear suspensions over the glass slide so that it forms a thin layer and it is
the size of nickel or larger. NO COVER SLIP USED!
o
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Let the slide air-dry—totally. This step facilitates the fixing of the smear to the slide.
Heat-fix the dry smear by running the slide quickly through the flame a few times. If
your fingers get hot, you have heat-fixed TOO MUCH. The heat-fixing will coagulate
some of the protein material and cause the suspension to adhere to the glass slide.
A different dye will be used for each bacterium–E. coli and crystal violet
Staphylococcus and safrinin
Staining the smear
o Place the slide on the wire mesh sitting over the dye tub
o Flood the smear with the dye: let sit for 1 minute.
Wash the slide WELL with distilled water. Blot the smear slide with your bibulous
paper pad.
Refer to the lab exercise on MICROSCOPY for help with the microscope.
Focus on the sample using the 10X lens (Be SURE that you are on brightfield
microscopy).
Focus on the smear using
the 100X oil immersion lens.
Be sure to read the
directions in the
MICROSCOPY exercise so
that you know how to move
to the 100X objective lens
using oil.
Identify the various shapes
and arrangements of
bacteria using the fields
below.
CLEAN YOUR SLIDES
using the slide cleanser at
the sinks.
2
LABORATORY REPORT SHEET
QUESTIONS:
1. Record your results of staining.
E. coli
Staphylococcus
2. Since everything on the slide will be the same color in simple staining, what you
possibly tell about the organisms?
3. Are all bacteria the same size?
4. What names are given to the shapes seen in the 2 bacteria used in lab?
5. Why heat-fix the smear?
6. Why are basic dyes more useful than acidic dyes when staining bacteria?
7. What is the disadvantage of having a really thick smear when staining?
3
GRAM STAIN TECHNIQUE
Page 1 of 6
Overview
The Gram stain procedure was originally developed by the Danish physician Hans Christian Gram to differentiate
pneumococci from Klebsiella pneumonia. In brief, the procedure involves the application of a solution of iodine
(potassium iodide) to cells previously stained with crystal violet or gentian violet. This procedure produces “purple
colored iodine-dye complexes” in the cytoplasm of bacteria. The cells that are previously stained with crystal violet
and iodine are next treated with a decolorizing agent such as 95% ethanol or a mixture of acetone and alcohol. The
difference between Gram-positive and Gram-negative bacteria is in the permeability of the cell wall to these “purple
colored iodine-dye complexes” when treated with the decolorizing solvent. While Gram-positive bacteria retain purple
iodine-dye complexes after the treatment with the decolorizing agent, Gram-negative bacteria do not retain complexes
when decolorized. To visualize decolorized Gram-negative bacteria, a red counter stain such as safranin is used after
decolorization treatment
Appearance of the Gram positive coccus and Gram negative bacillus at different stages of the gram staining procedure
are illustrated below:
Gram Kleuring.doc
November 7, 2001
Protocol
Molecular
Cell Physiology
GRAM STAIN TECHNIQUE
Page 2 of 6
Preparation of the smear
The first consideration is the correct preparation of the smear. Make a thin film of the material on a clean glass slide,
using a sterile loop or swab for viscous specimens. Air dry, then heat fix the slide by passing it several times through
a flame (the slide should not become too hot to touch). Failure to follow these directions may cause staining artifacts
and disrupt the normal morphology of bacteria and cells.
To be visible on a slide, organisms that stain by the Gram method must be present in concentrations of a minimum of
104 to 105 organisms/ml of unconcentrated staining fluid. At lower concentrations, the Gram stain of a clinical
specimen seldom reveals organisms even if the culture is positive. Smears that are not properly fixed tend to be
washed away during staining and washing resulting in the absence of stained bacteria.
In special situations, the following guidelines may be helpful:
When cerebrospinal fluid contains only a few organisms, they are more likely to be found if a concentrated “thick
smear” is examined. To prepare a “thick smear” the specimen is spun at high speed and a large drop of sediment (or
multiple drops, drying in between each drop) is placed in the center of the slide and allowed to air dry. The
cytocentrifuge may prove to be useful in concentrating bacteria as well as in preserving cell morphology.
When fluid specimens such as urine or CSF seem to vanish after the staining procedure, a wax mark, placed near the
smeared area on the slide (same side) after the staining procedure (to avoid introducing wax artifacts) will reduce
frustration in locating the specimen under the microscope. The wax mark can be used for quick focussing.
In a grossly bloody specimen, it may prove difficult to distinguish microorganisms from artifacts. After air-drying and
heat-fixing this type of specimen, the added preparatory step of covering it with distilled water, waiting five minutes,
and then rinsing, may cause the red blood cells to lyse and float off.
Gram Kleuring.doc
November 7, 2001
Protocol
Molecular
Cell Physiology
GRAM STAIN TECHNIQUE
Page 3 of 6
Staining procedure
1.
2.
3.
Flood slide with crystal (or gentian) violet- 60 seconds.
Flood with Gram’s iodine – 180 seconds.
Carefully decolorize with 95% ethanol until thinnest parts of the smear are colorless. (Wash with water).
This third step is the most critical and also the one most affected by technical variations in timing and
reagents.
4.
5.
Flood with safranin (pink color) (10% Fuchsine) – 60 seconds. (Wash with water).
Air dry, or blot with absorbent paper.
Gram Kleuring.doc
November 7, 2001
Protocol
Molecular
Cell Physiology
GRAM STAIN TECHNIQUE
Page 4 of 6
Results
As shown below, organisms that retain the violet-iodine complexes after washing in ethanol stain purple and are
termed Gram-positive, those that lose this complex stain red from the safranin counter stain are termed Gramnegative.
Typical Gram stain
negative
positive
Mix
Gram Kleuring.doc
November 7, 2001
Protocol
Molecular
Cell Physiology
GRAM STAIN TECHNIQUE
Page 5 of 6
Relationship of Cell Wall Structure to the Gram Stain
Are the structural differences between the cell walls of gram-positive and gram-negative Bacteria responsible in any
way for the Gram stain reaction?
In the Gram stain, an insoluble crystal violet-iodine complex is formed inside the cell, and this complex is extracted by
alcohol from gram-negative but not from gram-positive Bacteria. The alcohol dehydrates Gram_positive Bacteria,
which have very thick cell walls consisting of several layers of peptidoglycan. This causes the pores in the walls to
close, preventing the insoluble crystal violet-iodine complex from escaping. In gram-negative Bacteria, alcohol readily
penetrates the lipid-rich outer layer, and the thin peptidoglycan layer also does not prevent solvent passage, thus, the
crystal violet-iodine complex is easily removed.
Schematic diagrams of gram-positive and gram-negative cell walls.
Electron micrograph showing the cell walls.
Gram Kleuring.doc
November 7, 2001
Protocol
Molecular
Cell Physiology
GRAM STAIN TECHNIQUE
Page 6 of 6
Scanning electron micrographs of gram-positive (Bacillus subtilis) and gram-negative (Escherichia coli) bacteria. Note
the surface texture in the cells shown.
Gram-positive cell wall
Gram-negative cell wall.
Result of the Gram stain method
Gram Kleuring.doc
November 7, 2001
Protocol
Molecular
Cell Physiology
Endospore Staining by Schaeffer –Fulton Method.
Principle: Endospore staining is a differential staining technique where the spore is
stained in a manner so that it can be distinguished from the vegetative part of the cell.
Spores are structures remarkably resistant to heat, radiation, chemicals and other agents
that are typically lethal to the organism. The heat-resistence of spores has been linked to
their high content of calcium and dipicolinic acid. Although sporulation is genetically
regulated, the event is initiated when the organism senses depletion of nutrients or during
unfavorable environmental conditions. During sporulation, a vegetative cell gives rise to
a new intracellular structure termed as endospore that is surrounded by an impermeable
layer called sporecoat. Complete transformation of a vegetative cell into a sporangium
and then into a spore requires 6-8 hours in most spore-forming species. An endospore
develops in a characteristics position within a cell, i.e. either central ,subterminal or
terminal .Once an endospore is formed in a cell, the cellwall disintegrates ,releases the
endospore that becomes an independent spore. Endospores may remain dormant for long
period of time. However, a free spore may return to its vegetative or growing state with
the return of favourable conditions. The spores are differentially stained by using special
procedures that help dyes penetrate the spore wall. An aqueous primary stain (Malachite
Green) is applied and steamed to enhance penetration of the impermeable spore cores.
Once stained, the endospores do not readily decolourize and appear green within red
cells.
Suitable organism: Bacillus subtilis
Procedure: 1. Make a smear of the organisms on separate grease-free slides
2 Air dry and heat fix the smear.
3. Flood the smears with malachite green solution (stain for the spore).
3. Heat the slide to steaming and continue steaming for 5 minutes adding
more stains to the smear from time to time. (a set of slides may be steamed
for 10 and 15 minutes as well)
4. Wash under gentle stream of tap water.
5. Counter stain with safranin for 30 secs.
6. Wash smear with distilled water.
7. Air dry and examine under microscope.
Dorner’s Method:
Procedure: 1) 1ml Carbol fuchsin + 1 ml culture
2) boiling for 5, 10 & 15 min.
3) one drop placed in a grease free slide with inoculating needle .
4) The drop mixed with nigrosin.
5) Smear made very quickly.
6) observed under microscope.
••• Staining Endospores •••
Due to the highly resistant nature of endospores, they are not easily penetrated by stains.
Thus, it is necessary to steam the stain into an endospore. The Schaeffer-Fulton method
is the most commonly used endospore staining technique, which uses Malachite green as
the primary stain. Once the endospore has absorbed the stain, it is resistant to
decolorization, but the vegetative cell is easily decolorized
with water (leaving the vegetative cells colorless). Finally, the vegetative cells are
counterstained with Safranin to aid in their visualization. When viewed under a
microscope, the endospores appear green, while the vegetative cells are red or pink. The
steps in the endospore staining technique are listed below.
1. Using aseptic technique, prepare a bacterial smear on a clean slide, air dry and gently
heat fix.
2. Prepare a boiling water bath.
3. Cover the slide with a piece of paper towel, and place on a staining rack over the
water bath.
4. Flood the paper towel on the slide with Malachite green (primary stain).
5. Steam the slide for five minutes.
6. Remove the slide from the water bath, and remove the paper towel from the slide.
7. Allow the slide to cool, and then rinse with deionized water until the water runs clear.
8. Pour off any excess water and apply Safranin (counterstain) for two minutes.
9. Rinse excess Safranin off with deionized water, and blot the slide dry with blotting
paper.
10. Examine the slide with a light microscope under oil immersion objective
THE ACID-FAST STAIN
Robert Koch was the first person to isolate and identify Mycobacterium tuberculosis
from a patient with tuberculosis. He developed a stain for the bacterium, although it was
not very effective for visualizing this slender bacillus. Paul Ehrlich is the first to
describe the acid-fast properties of the bacterium. In the 1890s, Friedrich Neelsen and
Franz Ziehl modified the stain by adding phenol (carbolic acid) and basic fuschin. The
name of the dye carbol fuschin comes from the phenol and basic fuschin ingredients of
the stain.
The ability of the bacteria to resist decolorization with ACID alcohol confers acid
fastness to the bacterium. Acid-fast bacteria, of which there are very few—the major
genus Mycobacterium, have a high concentration of mycolic acid, a lipid, in their walls.
Although difficult to stain, once the stain goes into the wall, the cell will not de-stain or
decolorize easily. The ability of the bacteria to resist decolorization with acid (1%)
alcohol confers acid -fastness to the bacterium. It is thought that the phenol in the
carbol fuschin facilitates the dye going into the waxy wall of the bacterium. Although
gram positive, acid-fast bacteria do not take the crystal violet into the wall well,
appearing very light purple rather than the deep purple of normal gram positive bacteria
(the waxy lipid in the acid-fast wall repels the aqueous crystal violet stain).
As in the spore stain, steam is used as a gimmick to get the carbol fuschin primary dye
to go into the wall. Once in, it will not come out: But the acid alcohol decolorizer WILL
take it out of the nonacid-fast wall since the primary dye does not bind strongly to the
cell wall. Nonacid-fast bacteria will also take up the carbol fuschin, but the acid alcohol
decolorizer will remove it from wall since the primary dye does not bind strongly to the
cell wall.
Acid-fastness is an uncommon characteristic
shared by the genera Mycobacterium and
Nocardia (weakly acid-fast). Because of this
feature, this stain is extremely helpful in
identification in diseases caused by acid-fast
bacteria, particularly tuberculosis and leprosy.
In addition, the stain is used to determine the
presence of AF bacteria from lung tissue in
patients undergoing antibiotic therapy.
Acid-fast bacteria
Nonacid-fast bacteria
OBJECTIVES:
Learn to perform the acid-fast stain.
Differentiate between acid-fast and nonacid-fast bacteria.
MATERIALS NEEDED:
dye kit
stain rack
hot plate and beaker
paper towel (cut the size of the slide)
cultures: Mycobacterium and E. coli
THE PROCEDURE (Ziehl-Neelsen method):
1. Prepare the bacterial smears—air-dry, and heat-fix. (or the Mycobacterium
smear may be given to you already air-dried on a slide.)
2. Put a beaker of water on the hot plate and boil until steam is coming up from the
water. Then turn the hot plate down so that the water is barely boiling.
3. Place the wire stain rack over the beaker which now has steam coming up from
the boiled water.
4. Cut a small piece of paper towel and place it on top of the smear on the
slide. The towel will keep the dye from evaporating too quickly, thereby giving
more contact time between the dye and the bacterial walls.
5. Flood the smear with the primary dye, carbol fuschin, and leave for 5 minutes.
Keep the paper towel moist with the carbol fuschin. DO NOT let the dye dry on
the towel.
6. Remove the piece of paper towel and discard in the garbage. Wash the slide
really WELL with water.
7. Add the decolorizer acid alcohol (1% HCl + ethanol) and decolorize for 15-20
seconds. (The slide can be held, titled at an angle, and decolorizer dripped
down the slide.)
8. Wash WELL with water.
9. Flood the smear with the counterstain dye, methylene blue, and leave for 1
minute.
10. Rinse well with water. Blot dry with bibulous paper.
ALTERNATE COLD METHOD ACID FAST STAIN (Kinyoun)
The only difference is that without heat you have to stain the
smear with carbol fuschin for 10 minutes.
INTERPRETATION:
Acid-fast bacteria are hot pink or fuschia. Nonacid-fast bacteria are light blue.
QUESTIONS:
1. What is chemically unique about the Mycobacterium genus that causes it to be
acid-fast?
2. How is this stain procedure similar to the spore stain procedure?
Fall 2018 – Jackie Reynolds, Richland College, BIOL 2421

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