3.1: Introduction

Learning Objectives

  • Learn the differences between simple staining and differential staining techniques.
  • Learn how to prepare a bacterial smear from cultured organisms.
  • Learn the differences between gram positive and gram negative bacteria.
  • Learn how to perform the gram stain procedure.
  • Use microscopy to examine gram stained cells.
  • Learn about some special staining procedures, and view examples of these under oil immersion.

Most types of cells do not have much natural pigment and are consequently difficult to see under the light microscope unless they are stained. several types of stains are used to make bacterial cells more visible. In addition, specific staining techniques can be used to determine the cells ’ biochemical or structural properties, such as cell wall type and presence or absence of endospores. This type of data can help scientists identify and classify microorganisms, and can be used by health care providers to diagnose the cause of a bacterial infection .
One type of staining routine that can be used is the simple stain, in which entirely one stain is used, and all types of bacteria appear as the semblance of that stain when viewed under the microscope. Some stains normally used for simpleton staining include crystal violet, safranine, and methylene group blue. childlike stains can be used to determine a bacterial species ’ morphology and agreement, but they do not give any extra information .
Scientists will frequently choose to perform a differential stain, as this allows them to gather extra information about the bacteria they are working with. Differential stains use more than one stain, and cells will have a different appearance based on their chemical or structural properties. Some examples of differential gear stains are the Gram mark, acid-fast stain, and endospore mark. In this lab you will learn how to prepare bacterial cells for tarnish, and learn about the gram stain proficiency.

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The Gram Stain

This very normally used staining operation was first developed by the danish bacteriologist Hans Christian Gram in 1882 ( published in 1884 ) while working with weave samples from the lungs of patients who had died from pneumonia. Since then, the Gram mark procedure has been widely used by microbiologists everywhere to obtain important information about the bacterial species they are working with. Knowing the Gram reaction of a clinical isolate can help the health care professional make a diagnosis and choose the appropriate antibiotic for discussion .
Gram tarnish results reflect differences in cell wall constitution. Gram positive cells have thick layers of a peptidoglycan ( a carbohydrate ) in their cell walls ; Gram negative bacteria have identical little. Gram positive bacteria besides have teichoic acids, whereas Gram negatives do not. Gram negative cells have an out membrane that resembles the phospholipid bilayer of the cell membrane. The out membrane contains lipopolysaccharides ( LPS ), which are released as endotoxins when Gram negative cells die. This can be of concern to a person with an infection caused by a gram negative organism .
design 3.1.1 shows the major differences between the Gram positive and Gram negative cell walls ( besides refer to your casebook for extra data ). The differences in the cellular telephone wall constitution are reflected in the way the cells react with the stains used in the Gram stain procedure .
Gram stains are best performed on fresh cultures—older cells may have damaged cellular telephone walls and not give the proper Gram reaction. besides, some species are known as Gram-variable, and thus both Gram positive and Gram minus reactions may be visible on your chute .
Although the huge majority of bacteria are either Gram positive or Gram negative, it is important to remember that not all bacteria can be stained with this operation ( for model, Mycoplasmas, which have no cell wall, stain ailing with the Gram tarnish ).

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gram positive negative.pngFigure 3.1.1: Gram positive and Gram negative cell walls

Special Stains

There are a assortment of staining procedures used to identify specific external or inner structures that are not found in all bacterial species ( see board at the end of this exercise for a comparison of staining procedures ). You will do some of these staining procedures in the adjacent lab ( acid-fast stain and endospore staining ). In today ’ s lab, you will observe prepare slides of special stains : a capsule blot ( Klebsiella pneumoniae ), flagellum stain ( Proteus vulgaris ) and spirochete stain ( Treponema pallidum ) .

Capsule Stain

Some bacteria secrete a polysaccharide-rich social organization external to the cell wall called a glycocalyx. If the glycocalyx is dilute and broadly attached, it is called a slime layer ; if it is compact and tightly bound to the cell, it is called a capsule. The glycocalyx can protect the cell from dehydration and can allow the cell to stick to surfaces like tissues in the consistency. They may besides provide cells with protective covering against detection and phagocytosis by immune cells and contribute to the formation of a biofilm : in this way a glycocalyx can act as a virulence factor ; ( contributes to the ability of an organism to cause disease ) .
Capsules can be detected using a negative staining routine in which the background ( the slide ) and the bacteria are stained, but the space capsule is not stained. The encapsulate appears as a clean unstained zone around the bacterial cellular telephone. Since capsules are destroyed by heat, the capsule staining operation is done without heat-fixing the bacteria .

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Silver Stain

Flagella ( long whip-like structures used for bacterial motion ) and some bacteria ( e.g. spirochetes ) are excessively sparse to be observed with regular staining procedures. In these cases, a silver stain is used. Silver nitrate is applied to the bacteria along with a particular mordant ; the silver nitrate precipitates around the scourge or the sparse bacteria, thus thickening them so they can be observed under the light microscope .

Key Terms

Gram stain, bacterial smear, simple mark, differential stain, Gram positive, Gram negative, Gram variable, ejection seat, spirochete, scourge, negative staining, ash grey blot