PROCEDURE SIMPLE STAIN 1) Perform a bacterial smear of the given bacterial culture 2) Allow the smear to dry thoroughly. 3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three (2022)

Introduction to Bacterial Stains

Most bacteria are difficult to see under the bright field of a microscope. Bacteria are almost colorless (composed primarily of water) and therefore show little contrast with the suspension. To visualize bacteria, either dyes or stains are used. Since staining of bacterial cells is relatively fast, inexpensive, and simple, it is the most commonly used technique to visualize bacterial cells. Staining not only makes bacteria more easily seen, but it allows their morphology (e.g. size and shape) to be visualized more easily. In some cases, specific stains can be used to visualize certain structures (flagella, capsules, endospores, etc). of bacterial cells.

There are several staining methods that are used routinely with bacteria. These methods may be classified as 1) simple and 2) differential.Simple stains will react with all microbes in an identical fashion. They are useful solely for increasing contrast so that morphology, size, and arrangement of organisms can be determined. Differential stains give varying results depending on the organism being treated. These results are often helpful in identifying the microbe.

Commonly used microbiological stains generally fall into one of two categories - basic stains or acidic stains (although there are a few stains such as India Ink) which are neutral). A basic dye is a stain that is positively charged and will therefore react with material that is negatively charged. Bacterial cells have a slight negative charge will therefore attract and bind with basic dyes. Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue. Acid dyes are negatively charged and are repelled by the bacterial surface forming a deposit around the organism. They stain the background and leave the microbe transparent. Nigrosine and congo red are examples of acid dyes.

At first glance, the easiest way to stain bacterial cells would appear to be simply mixing the bacterial suspension with the dye and making a wet mount of this mixture. Unfortunately, if you were to try staining bacterial cells in this manner you would find that there was too much background (unbound dye) to allow for visualization of the cells. Therefore, you need to remove the unbound dye. Simply washing off the dye would result in removal of the cells along with the excess dye. Therefore, you need a mechanism to fix(adhere) the cells to the slide before staining to allow for removal of excess dye while keeping the cells on the slide.

A simple method is that of air drying and heat fixing. The organisms are heat fixed by passing an air-dried smear of the organisms through the flame of a gas burner. The heat coagulates the organisms' proteins causing the bacteria to stick to the slide. Be very careful not to over heat the organisms when fixing them to a slide. This distorts the sample of the organisms. Keep in mind the analogy of a fried egg. When you drop a raw egg onto a cold frying pan, it has a certain shape. Start heating it and the proteins (albumin) on the lower surface of the egg precipitate and fix the egg to the pan. At this point there has been a minimal distortion in the shape of the egg as a whole; only a small percent of the proteins have been precipitated. If you keep applying heat, the shape of the entire egg will change and eventually it will be reduced to charred remains. When you heat fix a slide, you want to apply enough heat to precipitate the proteins to allow the cells to stick to the slide but not to drastically change the shape of the cells (or reduce them to charred remains).

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (1)
Figure 2.1 Cocci bacteria stained blue

Simple Stain

Staining is a technique that is used to enhance contrast in a microscopic image. Staining can also be used to highlight structures for viewing. The simple stain can be used to determine cell shape, size, and arrangement. The simple stain is a very simple staining procedure involving only one stain. The most common stains (dyes) used are methylene blue, Gram safranin, and Gram crystal violet.

Basic stains, such as methylene blue, Gram safranin, or Gram crystal violet are useful for staining most bacteria.These stains will readily give up a OH- ion or accept a H+ ion, which leaves the stain positively charged.These positively charged stains adhere readily to the cell surface, since the surface of most bacteria are negatively charged.

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (2)
Figure 2.2 Bacillus simple stain

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (3)

Figure 2.3Bacteria cell before simple stain

Figure 2.4Bacteria cell after simple stain

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (4)

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (5)

Figure 2.5Bacteria cell before simple stain

Figure 2.6Bacteria cell after simple stain

VIRTUAL LAB

**Go to the site listed below to access a virtual lab in which you will perform a simple stain.

VIRTUAL SIMPLE STAIN

SKETCH 1
**Sketch a few of the bacteria you have viewed in the microscope, indicating any bacterial or background coloration you observe either with colored markers (pens, pencils, etc) or by labeling what colors you observe. Be sure to include a few of each bacteria if they have stained differently.

Click Here to Access a Chart to Produce Your Sketches (MS WORD)

Click Here to Access a Chart to Produce Your Sketches (PDF)

Endospore Stain

(Video) MICROBIOLOGY | Bacterial Staining (Smear Preparation & Simple Staining)

The endospore stain is a differential stain used to visualize bacterial endospores.Endospores are formed by some bacteria, such as Bacillus.By forming spores, bacteria can survive in hostile conditions.Spores are resistant to heat, dessication, chemicals, and radiation. Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions. The normally growing cell that forms the endospore is called a vegetative cell. Spores are metabolically inactive and dehydrated.They can remain viable for thousands of years.When spores are exposed to favorable conditions, they can germinate into a vegetative cell within 90 minutes.

Endospores can form within different areas of the vegetative cell. They can be central, subterminal, or terminal.Central endospores are located within the middle of the vegetative cell.Terminal endospores are located at the end of the vegetative cell.Subterminal endospores are located between the middle and the end of the cell. Endospores can also be larger or smaller in diameter than the vegetative cell. Those that are larger in diameter will produce an area of "swelling" in the vegetative cell.These endospore characteristics are consistent within the spore-forming species and can be used to identify the organism.

Because of their tough protein coats made of keratin, spores are highly resistant to normal staining procedures. The primary stain in the endospore stain procedure, malachite green, is driven into the cells with heat and readily adheres to the endospore. Since malachite green is water-soluble and does not adhere well to the cell, and since the vegetative cells have been disrupted by heat, the malachite green rinses easily from the vegetative cells, allowing them to readily take up the counterstain. This allows the endospores to be visible with the malachite green stain and the vegetative cells to be visible with the counterstain.

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (6)
Figure 2.7Bacilli endospores stained green and vegetative cells stained red

PROCEDURE ENDOSPORE STAIN
1) Perform a bacterial smear of Bacillus or the organism you are given to stain
2) Saturate the smear with malachite green
3) Heat the slide gently over a Bunsen burner for 5 minutes
4) Rinse the slide gently with water
5) Counterstain with safranin for 2 minutes
6) Rinse the slide gently with water
7) Observe the slide under the microscope. Endospores will stain green. Vegetative cells will stain red

VIRTUAL LAB

**Go to the site listed below to access a virtual lab in which you will perform an endospore stain.

VIRTUAL

ENDOSPORE STAIN

SKETCH 2
**Sketch a few of the bacteria you have viewed in the microscope, indicating any bacterial or background coloration you observe either with colored markers (pens, pencils, etc) or by labeling what colors you observe. Be sure to include a few of each bacteria if they have stained differently.

Click Here to Access a Chart to Produce Your Sketches (MS WORD)

Click Here to Access a Chart to Produce Your Sketches (PDF)

Capsule Stain

The capsule stain employs an acidic stain and a basic stain to detect capsule production. Capsules are formed by organisms such as Klebsiella pneumoniae. Most capsules are composed of polysaccharides, but some are composed of polypeptides. The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. Some capsules have well-defined boundaries, and some have fuzzy, trailing edges. Capsules protect bacteria from the phagocytic action of leukocytes and allow pathogens to invade the body. If a pathogen loses its ability to form capsules, it can become avirulent.

Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic stain and to stain the cell itself using a basic stain. We will use India ink and Gram crystal violet. This leaves the capsule as a clear halo surrounding a purple cell in a field of black.

The medium in which the culture is grown as well as the temperature at which it is grown and the age of the culture will affect capsule formation. Older cultures are more likely to exhibit capsule production. When performing a capsule stain on your unknown, be sure the culture you take your sample from is at least five days old.

The ability to produce a capsule is an inherited property of the organism, but the capsule is not an absolutely essential cellular component. Capsules help many pathogenic and normal flora bacteria to initially resist phagocytosis by the host's phagocytic cells. In soil and water, capsules help prevent bacteria from being engulfed by protozoans. Capsules also help many bacteria to adhere to surfaces and thus resist flushing. It also enables many bacteria to form biofilms. Abiofilm consists layers of bacterial populations adhering to host cells and embedded in a common capsular mass.

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (7)
Figure 2.8Bacilli capsule stain

PPROCEDURE CAPSULE STAIN

1)Place a single drop ofIndia inkon a microscope slide

2)Add the organism to be stained and mix into the drop ofIndia ink
3)Spread the mixture on the slide to a thin layer

4)Allow the film to air dry.DO NOTheat or blot dry. Heat will melt the capsule

5)Saturate the slide withcrystal violetfor 1 minute

6)Rinse the slide gently with water

(Video) ONLINE Micro Lab 4: Bacterial Structure, Simple Stains, Negative Stains, Gram & Acid-Fast Stains

7)Allow the slide to air dry
8)Observe the slide under the microscope.The background will be dark. The bacterial cells will be stained purple. The capsule (if present) will appear clear against the dark background

VIRTUAL LAB

**Go to the site listed below to access a virtual lab in which you will perform a capsule stain

VIRTUAL

CAPSULE STAIN

SKETCH 3
**Sketch a few of the bacteria you have viewed in the microscope, indicating any bacterial or background coloration you observe either with colored markers (pens, pencils, etc) or by labeling what colors you observe. Be sure to include a few of each bacteria if they have stained differently.

Click Here to Access a Chart to Produce Your Sketches (MS WORD)

Click Here to Access a Chart to Produce Your Sketches (PDF)

Acid Fast Stain

The acid-fast stain is a differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium.Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids. Acid-fast organisms are highly resistant to disinfectants and dry conditions.

Because the cell wall is so resistant to most compounds, acid-fast organisms require a special staining technique.The primary stain used in acid-fast staining, carbolfuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall.This is further assisted by the addition of heat.The smear is then rinsed with a very strong decolorizer, which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms. The decolorized non-acid-fast cells then take up the counterstain.

The acid-fast stain is an especially important test for the genus Mycobacterium. There are two distinct pathogens in this group: M. tuberculosis, the causative organism of tuberculosis, and M. leprae, the causative agent of leprosy.

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (8)
Figure 2.9Acid Fast stain
(A) non-acid fast bacteria, (B) acid fast bacteria

PPROCEDURE ACID FAST STAIN

1)Perform a bacterial smear
2)Flood the smear withcarbolfuchsin

3)Heat the slide gently over the Bunsen burner for 5 minutes

4)Rinse the slide gently with water

5)Decolorize the slide with acid-alcohol until the rinsate runs clear

6)Rinse the slide gently with water

7)Counterstain withmethylene bluefor 2 minutes

8)Rinse the slide gently with water

9)Observe the slide under the microscope.Acid-fast cells will stain fuchsia (pink or red). Non-acid-fast cells will stain blue

VIRTUAL LAB

**Go to the site listed below to access a virtual lab in which you will perform an acid fast stain

VIRTUAL

ACID FAST STAIN
(Video) Lab 3-10: Endospore Stain

SKETCH 4
**Sketch a few of the bacteria you have viewed in the microscope, indicating any bacterial or background coloration you observe either with colored markers (pens, pencils, etc) or by labeling what colors you observe. Be sure to include a few of each bacteria if they have stained differently.

Click Here to Access a Chart to Produce Your Sketches (MS WORD)

Click Here to Access a Chart to Produce Your Sketches (PDF)

Negative Stain

The negative stain is particularly useful for determining cell size and arrangement. It can also be used to stain cells that are too delicate to be heat-fixed. We will use nigrosin as our negative stain.

Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion and becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background.

Negative staining is an excellent way to determine an organism’s cellular morphology. Since the cells themselves are not stained, their morphology is not distorted in any way. The nigrosin provides a dark background against which the shapes of the unstained cells are clearly visible. This method provides a high degree of contrast not available in most other staining procedures.

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (9)
Figure 2.10Negative Stain

PPROCEDURE NEGATIVE STAIN

1)Place a single drop ofnigrosinon a microscope slide

2)Mix a sample of your organism to stain into the drop ofnigrosin

3)Spread the sample on the slide into a thin layer
4)
Allow the film to air dry

5)Observe the slide under the microscope. The bacteria will be clear and the background will stain dark

VIRTUAL LAB

**Go to the site listed below to access a virtual lab in which you will perform a negative stain

VIRTUAL NEGATIVE

STAIN

SKETCH 5
**Sketch a few of the bacteria you have viewed in the microscope, indicating any bacterial or background coloration you observe either with colored markers (pens, pencils, etc) or by labeling what colors you observe. Be sure to include a few of each bacteria if they have stained differently.

Click Here to Access a Chart to Produce Your Sketches (MS WORD)

Click Here to Access a Chart to Produce Your Sketches (PDF)

Gram Stain

The Gram stain is the most important staining procedure in microbiology. It is used to differentiate between gram positive organisms and gram negative organisms. Hence, it is a differential stain. Gram negative and gram positive organisms are distinguished from each other by differences in their cell walls. These differences affect many aspects of the cell, including the way the cell takes up and retains stains.

(Video) Introduction to Microbiology Culture Techniques

Gram positive cells take up crystal violet, which is then fixed in the cell with iodine mordant. This forms a crystal-violet iodine complex which remains in the cell even after decolorizing.It is thought that this happens because the cell walls of gram positive organisms include a thick layer of protein-sugar complexes called peptidoglycans. This layer makes up 60-90% of the gram positive cell wall. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell.At the end of the gram staining procedure, gram positive cells will be stained a purplish-blue color.

Gram negative cells also take up crystal violet, and the iodine forms a crystal violet-iodine complex in the cells as it did in the gram positive cells. However, the cell walls of gram negative organisms do not retain this complex when decolorized. Peptidoglycans are present in the cell walls of gram negative organisms, but they only comprise 10-20% of the cell wall. Gram negative cells also have an outer layer which gram positive organisms do not have; this layer is made up of lipids, polysaccharides, and proteins. Exposing gram negative cells to the decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. This allows the cells to subsequently be stained with safranin.At the end of the gram staining procedure, gram negative cells will be stained a reddish-pink color.

Often, detecting the presence of microorganisms and determining whether an infection is caused by an organism that is Gram-positive or Gram-negative will be sufficient to allow a doctor to prescribe treatment with an appropriate antibiotic while waiting for more specific tests, such as a culture, to be completed.

PROCEDURE SIMPLE STAIN1) Perform a bacterial smear of the given bacterial culture2) Allow the smear to dry thoroughly.3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times.Overheating can distort the cells.4) Saturate the smear with basic dye and let sit for approximately 1 minute. You may use crystal violet, safranin, or methylene blue5) Rinse the slide gently with water6) Observe the slide under the microscope (10)
Figure 2.11Gram Stain

PROCEDURE

1)Perform a bacterial smear of your given culture
2)Saturate the smear withcrystal violetfor 1 minute

3)Rinse the slide gently with water

4)Saturate the smear withiodinefor 1 minute

5)Rinse the slide gently with water

6)Decolorize with Gram decolorizer (acetone/alcohol) for 3-5 seconds

7)Rinse the slide gently with water

8)Counterstain withsafraninfor 1 minute

9)Rinse the slide gently with water

10)Observe the slide under the microscope.Gram positive bacteria will stain purple. Gram negative bacteria will stain red/pink

VIRTUAL LAB

**Go to the site listed below to access a virtual lab in which you will perform a gram stain

VIRTUAL GRAM

STAIN

SKETCH 6
**Sketch a few of the bacteria you have viewed in the microscope, indicating any bacterial or background coloration you observe either with colored markers (pens, pencils, etc) or by labeling what colors you observe. Be sure to include a few of each bacteria if they have stained differently.

Click Here to Access a Chart to Produce Your Sketches (MS WORD)

Click Here to Access a Chart to Produce Your Sketches (PDF)

QUESTIONS

**Answer the questions on bacterial staining given below

1)

(Video) Lab 3 Smear prepration and simple staining

While performing the stains, why was it important to not let the slide get too hot from the bunsen burners?
2)
How does a simple stain differ from a negative stain?
3)
What is the difference between a simple stain and a differential stain?
4)
What does "fixing" mean when performing bacterial stains?
5)
How are positively charged dyes able to adhere to bacteria?
6)
What survival advantage is gained in bacteria that produce capsules?
7)
How long can endospores survive?
8)
What survival advantage do acid fast bacteria have over other non-acid fast bacteria?
9)
What variety of stain is negatively charged rather than positively charged?
10)
List some differences in gram negative and gram positive bacterial cell walls.

Click Here for an MS WORD Version of the Bacterial Stain Questions

Click Here for a PDF Version of the Bacterial Stain Questions

FAQs

What is the procedure for a simple stain? ›

What is simple staining of bacteria? ›

Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail, in contrast to negative staining where the bacteria remain unstained against a dark background.

What is the purpose of the simple stain procedure? ›

Simple staining allows one to observe the morphology and arrangement of the bacterial cells. Morphology refers to “form” or shape. We can use terms like spiral, bacilli (rod shaped), and cocci (spherical) to describe cell shapes.

What is the purpose of simple staining a bacterial smear? ›

The purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells. The most commonly used basic stains are methylene blue, crystal violet, and carbol fuchsin.

Videos

1. MICROBIOLOGY PART 2: Gram stain
(A Level Biology: Mr Ashcroft)
2. BIOL 2P98 2018 - part 1, introduction to your microbiology lab
(Microbiology Videos)
3. Tutorial - Gram Staining
(Dr. C. Shane Ramey)
4. The USFWS Guidebook to Salmon Disease in Fish (Salmonid Diseases)
(Free Nature Interviews)
5. Introduction to microbiology and culture techniques
(Vidya-mitra)
6. Microscope Intro
(Andrew Vinal)

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