Report of Microbiology Experiment
Smears, Simple Staining, Gram Staining and Structural Stains
XXX 2012/3/25 Shandong University
Purpose
1. Understand the rationale and procedure of all the staining methods.
2. Perform and interpret all the stains.
3. Learn the shapes and structures of bacteria in deep.
Background
1. Smear Preparation
Smear is to place a thin film of bacterial cells on a slide. The smear must be fixed to kill the bacteria; coagulated proteins from the cells will cause cells to stick to the slide. Fixing denatures bacterial enzymes, preventing them from digesting cell parts, which cause the cell to break. Fixing also preserves microbes with minimal shrinkage or distortion when stained.
2. Simple Stain
Staining procedures use only one stain are called simple stains. The dyes are usually salts composed of charged colored ions. If the chromophore is a positive ion, the stain is considered a basic stain; if it is a negative ion, it is an acidic stain. Most bacteria are stained when a basic stain permeates the cell wall and adheres by weak ionic bonds to the bacterial cell, which is slightly negatively charged.
3. Gram Stain
Gram stain is a stain that classifies bacteria as either gram-positive or gram-negative.
Bacteria differ in their rate of decolorization. Those decolorize easily are referred to as gram-negative, whereas those that decolorize slowly and retain the primary stain are called gram-positive. This difference is because of the chemical and physical differences in bacteria’s cell wall. Gram-positive cell walls contain multiple layers of peptidoglycans which gram-negative cells only have a thin layer. When decolorized by alcohol, the crystal violet in gram-positive cells cannot be washed out because of the compact peptidoglycan. So there will be different colors for different bacteria. When bacteria die, their cell walls degrade and may not retain the primary stain, giving inaccurate results.
4. Structural Stains
Structural stains can be used to identify and study the structure of bacteria when
there is no electron microscope.
Endospores are “resting bodies” of several genera of bacteria. They do not
metabolize and are resistant to heating, various chemicals, and many harsh environmental conditions. To know whether a bacterium is an endospore-former and also the position of the endospores is helpful. Endospores are impermeable to most stains, so heat is usually applied to drive the stain into the endospore. Once stained, the endospores do not readily decolorize.
Flagella are thin proteinaceous structures that originate in the cytoplasm and
project out from the cell wall. They are very frafgile (usually 20-50nm) and are
not visible with a light microscope. But when they are coated with a mordant, which increases their diameter, they can be seen with light microscopes.
The presence and location of fragella are helpful in identifying and classifying
bacteria. There are two main types of fragella: peritrichous and polar.
Materials
Compound light microscopes
Slides
Inoculating loop
Alcohol burner
Absorbent paper
Methylene blue(simple stain)
Gram-staining reagents(Crystal violet, Gram’s iodine, Ethanol, Safranin)
Endospore stain reagents(Malachite green and Safranin)
Flagella stain reagents(A and B)
Distilled water
Cultures
Escherichia coli slant
Staphylococcus aureus slant
Bacillus subtilis slant(24h&72h)
Clostridium sporogenes slant(24h&72h)
Pseudomonas aeruginosa slant
Procedure
ⅠPreparing smears
1. Clean the slides.
2. Put a drop of distilled water on a slide.
3. Sterilize the inoculating loop.
4. Using the cooled loop, scrape a small amount of the culture off the slant and emulsify the cells in the drop of water.
5. Let the smears dry.
6. Pass the slide quickly through the blue flame with smear side up two or three times.
ⅡSimple Stain(E.coli , S.aureus and B.subtilis)
1. Prepare smears.
2. Cover the smears with Methylene blue and leave them for about 1min.
3. Gently wash off the Methylene blue with water.
4. Blot the slides dry and examine stained smears microscopically.
ⅢGram Stain(E.coli , S.aureus and B.subtilis)
1. Prepare fixed smears.
2. Cover the smears with crystal violet for 1-2 min.
3. Gently wash off the Crystal violet with water.
4. Cover the smears with Gram’s iodine for 1 min.
5. Gently wash off the Gram’s iodine with water.
6. Decolorize the smears with ethanol.
7. Gently wash off the ethanol.
8. Cover the smears with safranin for 2 min.
9. Gently wash off the safranin.
10. Blot the slides dry and examine stained smears microscopically.
ⅣStructural Stains
The endospore stain(B.subtilis and C.sporogenes (24h&72))
1. Prepare fixed smears of bacteria.
2. Place a piece of absorbent paper over the smears.
3. Cover the paper with Malachite green.
4. Steam the slides for 5 min.
5. Wash the smears with water.
6. Cover the smears with Safranin for 2 min.
7. Wash the smears with water and blot them dry.
8. Examine stained smears with a microscope.
The flagella stain(B.subtilis , E.coli and P .aeruginosa )
1. Place a drop of distilled water on a side of a slide.
2. Lightly touch the drop of water with the bacteria without touching the slide.
3. Tilt the slide so the drop will flow to the opposite side of the slide.
4. Allow the slide to air dry.
5. Cover the dried smear with dye liquor A for 5 min.
6. Gently wash off the dye liquor A with distilled water.
7. Gently wash the slide with dye liquor B until brown color appears.
8. Wash the smears with water and blot them dry.
9. Examine stained smears with a microscope.
Results
Simple stain
E.coli 1000×
S.aureus
1000×
B.subtilis 1000×
Gram stain
E.coli and S.aureus 1000×
Unknown bacteria 1000× Gram positive & gram negative bacillus
Unknown bacteria 1000× Gram negative bacillus
Endospore stain
B.subtilis 1000× Central, swollen endospores
C.sporogenes 1000× 24h Terminal, swollen endospores
C.sporogenes 1000× 72h Free endospores
Flagella stain
B.subtilis
1000× Peritrichous flagella
E.coli 1000× Peritrichous flagella
P .aeruginosa 1000× Monotrichous flagellum
Rethink
1. The microscopes should have a blue optical filter in the condenser to make sure the blank filed is white. That will be helpful to recognize the color of different stains.
2. Heat-fixing is important in smear preparation to keep the shape of cells. When performing flagella stains, I found that many bacterial cells looks larger than their normal size in the field. It may because of autolysis when cells die.
3. When performing a Gram stain, the time of decolorizing should be handle to prevent excessive decolorizing.
4. When performing flagella stains, violent shaking should be avoided to prevent the flagella from coming off. According to my results, bacteria with few flagellas will easily lose their flagellas when being stained.
此实验报告存在一部分小错误和疏漏，比如个别的语法错误、用词不当，裁剪的图片未标明比例尺等。仅供参考。
Report of Microbiology Experiment
Smears, Simple Staining, Gram Staining and Structural Stains
XXX 2012/3/25 Shandong University
Purpose
1. Understand the rationale and procedure of all the staining methods.
2. Perform and interpret all the stains.
3. Learn the shapes and structures of bacteria in deep.
Background
1. Smear Preparation
Smear is to place a thin film of bacterial cells on a slide. The smear must be fixed to kill the bacteria; coagulated proteins from the cells will cause cells to stick to the slide. Fixing denatures bacterial enzymes, preventing them from digesting cell parts, which cause the cell to break. Fixing also preserves microbes with minimal shrinkage or distortion when stained.
2. Simple Stain
Staining procedures use only one stain are called simple stains. The dyes are usually salts composed of charged colored ions. If the chromophore is a positive ion, the stain is considered a basic stain; if it is a negative ion, it is an acidic stain. Most bacteria are stained when a basic stain permeates the cell wall and adheres by weak ionic bonds to the bacterial cell, which is slightly negatively charged.
3. Gram Stain
Gram stain is a stain that classifies bacteria as either gram-positive or gram-negative.
Bacteria differ in their rate of decolorization. Those decolorize easily are referred to as gram-negative, whereas those that decolorize slowly and retain the primary stain are called gram-positive. This difference is because of the chemical and physical differences in bacteria’s cell wall. Gram-positive cell walls contain multiple layers of peptidoglycans which gram-negative cells only have a thin layer. When decolorized by alcohol, the crystal violet in gram-positive cells cannot be washed out because of the compact peptidoglycan. So there will be different colors for different bacteria. When bacteria die, their cell walls degrade and may not retain the primary stain, giving inaccurate results.
4. Structural Stains
Structural stains can be used to identify and study the structure of bacteria when
there is no electron microscope.
Endospores are “resting bodies” of several genera of bacteria. They do not
metabolize and are resistant to heating, various chemicals, and many harsh environmental conditions. To know whether a bacterium is an endospore-former and also the position of the endospores is helpful. Endospores are impermeable to most stains, so heat is usually applied to drive the stain into the endospore. Once stained, the endospores do not readily decolorize.
Flagella are thin proteinaceous structures that originate in the cytoplasm and
project out from the cell wall. They are very frafgile (usually 20-50nm) and are
not visible with a light microscope. But when they are coated with a mordant, which increases their diameter, they can be seen with light microscopes.
The presence and location of fragella are helpful in identifying and classifying
bacteria. There are two main types of fragella: peritrichous and polar.
Materials
Compound light microscopes
Slides
Inoculating loop
Alcohol burner
Absorbent paper
Methylene blue(simple stain)
Gram-staining reagents(Crystal violet, Gram’s iodine, Ethanol, Safranin)
Endospore stain reagents(Malachite green and Safranin)
Flagella stain reagents(A and B)
Distilled water
Cultures
Escherichia coli slant
Staphylococcus aureus slant
Bacillus subtilis slant(24h&72h)
Clostridium sporogenes slant(24h&72h)
Pseudomonas aeruginosa slant
Procedure
ⅠPreparing smears
1. Clean the slides.
2. Put a drop of distilled water on a slide.
3. Sterilize the inoculating loop.
4. Using the cooled loop, scrape a small amount of the culture off the slant and emulsify the cells in the drop of water.
5. Let the smears dry.
6. Pass the slide quickly through the blue flame with smear side up two or three times.
ⅡSimple Stain(E.coli , S.aureus and B.subtilis)
1. Prepare smears.
2. Cover the smears with Methylene blue and leave them for about 1min.
3. Gently wash off the Methylene blue with water.
4. Blot the slides dry and examine stained smears microscopically.
ⅢGram Stain(E.coli , S.aureus and B.subtilis)
1. Prepare fixed smears.
2. Cover the smears with crystal violet for 1-2 min.
3. Gently wash off the Crystal violet with water.
4. Cover the smears with Gram’s iodine for 1 min.
5. Gently wash off the Gram’s iodine with water.
6. Decolorize the smears with ethanol.
7. Gently wash off the ethanol.
8. Cover the smears with safranin for 2 min.
9. Gently wash off the safranin.
10. Blot the slides dry and examine stained smears microscopically.
ⅣStructural Stains
The endospore stain(B.subtilis and C.sporogenes (24h&72))
1. Prepare fixed smears of bacteria.
2. Place a piece of absorbent paper over the smears.
3. Cover the paper with Malachite green.
4. Steam the slides for 5 min.
5. Wash the smears with water.
6. Cover the smears with Safranin for 2 min.
7. Wash the smears with water and blot them dry.
8. Examine stained smears with a microscope.
The flagella stain(B.subtilis , E.coli and P .aeruginosa )
1. Place a drop of distilled water on a side of a slide.
2. Lightly touch the drop of water with the bacteria without touching the slide.
3. Tilt the slide so the drop will flow to the opposite side of the slide.
4. Allow the slide to air dry.
5. Cover the dried smear with dye liquor A for 5 min.
6. Gently wash off the dye liquor A with distilled water.
7. Gently wash the slide with dye liquor B until brown color appears.
8. Wash the smears with water and blot them dry.
9. Examine stained smears with a microscope.
Results
Simple stain
E.coli 1000×
S.aureus
1000×
B.subtilis 1000×
Gram stain
E.coli and S.aureus 1000×
Unknown bacteria 1000× Gram positive & gram negative bacillus
Unknown bacteria 1000× Gram negative bacillus
Endospore stain
B.subtilis 1000× Central, swollen endospores
C.sporogenes 1000× 24h Terminal, swollen endospores
C.sporogenes 1000× 72h Free endospores
Flagella stain
B.subtilis
1000× Peritrichous flagella
E.coli 1000× Peritrichous flagella
P .aeruginosa 1000× Monotrichous flagellum
Rethink
1. The microscopes should have a blue optical filter in the condenser to make sure the blank filed is white. That will be helpful to recognize the color of different stains.
2. Heat-fixing is important in smear preparation to keep the shape of cells. When performing flagella stains, I found that many bacterial cells looks larger than their normal size in the field. It may because of autolysis when cells die.
3. When performing a Gram stain, the time of decolorizing should be handle to prevent excessive decolorizing.
4. When performing flagella stains, violent shaking should be avoided to prevent the flagella from coming off. According to my results, bacteria with few flagellas will easily lose their flagellas when being stained.
此实验报告存在一部分小错误和疏漏，比如个别的语法错误、用词不当，裁剪的图片未标明比例尺等。仅供参考。