Basic Reagents and solutions for Microbiological Tests - labsstudies

Basic Reagents and solutions for Microbiological Tests

Basic Reagents and solutions for Microbiological Tests

Microbiological tests refer to group of tests used to determine and differentiate microorganisms that are medical importance. The Micro organisms that are medical importance are Bacteria, Fungi and Virus.

In this article you will learn:

  • Definitions of terms in Microbiological Tests: Microbiology, Microorganisms and Medical Microbiology.
  • Basic Reagents and solutions for Microbiological Tests
  • Principle, uses , components, advantages and disadvantages of each Reagents for Microbiological Tests

Definitions of terms in Microbiological Tests


  • Microbiology is the study of microorganisms.


  • Are organisms that cannot be seen by naked eyes e.g. bacteria, viruses and fungi.
  • Devices like microscope are needed for them to be seen. Microscope is used to examine this microorganisms , because they can not seen by our Naked eyes.

Medical Microbiology

  • Is the branch of microbiology which deal with the study of microorganisms of medical importance.

Basic Reagents and solutions for Microbiological Tests

  • Ziehl Neelsen Stain (ZN stain)
  • Gram Stain
  • Wayson’s Stain
  • KOH

Solutions in Microbiology

  • Normal saline
  • Sulphuric acid
  • HCl
  • Methanol
  • Wayson solution A&B
  • KOH


Principle, uses , components, advantages and disadvantages of each Reagents 

Ziehl Neelsen Stain (ZN stain)

Used to stain Mycobacterium species including;

  1. tuberculosis
  2. ulcerans
  3. leprae
  4. non-tuberculous mycobacteria (NTM).  
  • Used to stain also Cryptosporidium oocysts.
  • So it provides the initial bacteriologic evidence of the presence of Mycobacteria in a clinical specimen.
  • Smear microscopy is the quickest and easiest procedure that can be performed.

Basic Reagents and solutions for Microbiological Tests

Mycobacteria species stained by ZN stain: CDC

Components of Ziehl Neelsen Stain are:

  • 70% alcohol
  • Strong Carbol fuchsin
  • 20% Sulphuric acid and
  • 0.1% methylene blue

Principle of ZN stain

  • When Mycobacterium and Cryptosporidium oocysts are stained with hot, strong Carbol fuchsin, they resist decolorization with a solution of acid or acid/alcohol and stain red.
  • Tissues and other organisms are decolorized by the acid/alcohol solution and are demonstrated by a counterstain, e.g. methylene blue, which stain blue.
  • This property is due to the presence of a waxy substance in the cell wall called mycolic acid.

Advantages of using ZN Stain

  • Microscopy of sputum smears is simple and inexpensive.
  • quickly detecting infectious cases of pulmonary TB.
  • Sputum specimens from patients with pulmonary TB – especially those with cavitary disease – often contain sufficiently large numbers of acid-fast bacilli to be readily detected by microscopy.

Disadvantages of using ZN Stain

  • Direct smear microscopy is relatively insensitive as at least 5,000 bacilli per millilitre of sputum are required for direct microscopy to be positive.
  • Smear sensitivity is further reduced in patients with extra-pulmonary TB, those with HIV-co-infection, and those with disease due to nontuberculous mycobacteria (NTM).

Limitations of AFB microscopy

  • It cannot distinguish Mycobacterium tuberculosis from NTM.
  • It cannot distinguish viable from non-viable organisms.
  • It cannot distinguish drug-susceptible from drug-resistant strains.


The Gram Stain

  • Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884.
  • This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms.

Components of Gram stain are:

  • Crystal violet (primary stain)
  • Lugol’s iodine(mordant)
  • Acid in alcohol(decolorizer)
  • Neutral red(counter stain)

The Principle of Gram Stain

  • When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol.
  • The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. 
  • 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. 
  • So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in color.
  • In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off.
  • When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and appears red in color.

Advantages of Gram Stain

  • Helps in the classification and differentiations of microorganisms into Gram positive and gram negative.
  • It gives quick results when examining infections. i.e. culture vs gram stain.
  • It is simple and cost-effective.
  • It helps with determining appropriate treatments for infections.
  • It allows for various methods of testing.
  • It is basically a key procedure in identifying bacteria.

Disadvantages of Gram Stain

  • Needs proper monitoring of staining time, because over or under staining and over decolorizing affects the identification of the bacteria.
  • Bacteria are not categorized into species level.
  • It is not a single staining procedure.


Wayson’s Stain

  • Wayson’s staining is a rapid method which shows clearly the bipolar staining morphology of bacteria such as Yersinia pestis.

Components of Wayson’s Stain are:

  • It consists of Wayson reagent A&B.
  • Wayson Reagent A: Fuchsin and Methylene blue
  • Wayson Reagent B: 5% aqueous phenol

The principle of Wayson’s stain

  • The principle of Wayson’s stain (Fuchsin, methylene blue and aqueous phenol) is to provide contrasting staining so that bacteria, tissue and blood cell components are easily seen.
  • It is frequently used to detect Yersinia pestis in sputum smears.

Advantages of Wayson’s stain

  • It is frequently used to detect Yersinia pestis in sputum smears. For diagnosis of bubonic plague.
  • It is used along with the Giemsa and Wright’s stains to rapidly detect potential biowarfare attacks.
  • It has also been investigated as a possible cheaper and faster way to detect melioidosis.
  • It is a useful alternative to Gram stains, especially for detecting Yersinia enterocolitica which is often found in contaminated food.
  • It is rapid and simple to perform.


Potassium hydroxide (KOH)

  • It is used for the rapid detection of fungal elements in clinical specimen.
  • Dermatophyte-A type of fungus that causes diseases of the skin, including tinea or ringworm.
  • KOH – The chemical formula for potassium hydroxide, which is used to perform the KOH test. The tests is also called a potassium hydroxide preparation.
  • Thrush -A disease of the mouth, caused by Candida albicans and characterized by a whitish growth and ulcers. It can be diagnosed with the KOH test.
  • Tinea- A superficial infection of the skin, hair, or nails, caused by a fungus and commonly known as ringworm.


The Principle of KOH Preparation/test

  • KOH is a strong alkali. When specimen such as skin, hair, nails or sputum is mixed with 20% w/v KOH, it softens, digests and clears the tissues (e.g., keratin present in skins).
  • This enables the surrounding fungi to be easily seen under microscope.
  • Dermatophytes are easily recognized under the microscope by their long branch-like structures.
  • Yeast cells look round or oval.


Advantages of KOH

  • In diagnostic laboratories, KOH mount is one of the main methods of investigating fungal infections. 
  • It is used as a primary screening tool, it detects fungal elements present but may not necessarily identify the species of the fungi.

Note: To identify the fungal isolate, specimen must be cultured.

  • It is a simple and rapid method to diagnose fungal infections.

Disadvantages of KOH Preparation Method

  • Experience required since background artifacts are often confusing.
  • Clearing of some specimens may require an extended time.
  • Potassium hydroxide is a highly corrosive deliquescent chemical.

Preparation of KOH

Procedure to make 100 ml of KOH 20% w/v solution:

  • Weigh 20 g potassium hydroxide (KOH) pellets.
  • Transfer the chemical to a screw-cap bottle.
  • Add 50 ml distilled water, and mix until the chemical is completely dissolved, add remaining distilled water and make the volume 100 ml.
  • Label the bottle and mark it corrosive.
  • Store it at room temperature.
  • The reagent is stable for up to 2 years.

Read also: Basic reagents for Parasitological Tests



  • F.J. Baker, R.E. Silverton (2001): Introduction to Medical Laboratory Technology, 7thEdition, Oxford University Press; Oxford, England.
  • Monica Cheesbrough (1981): Medical Laboratory Manual for Tropical Countries Volume I, 2nd Edition Butterworth & Co. (Publishers) Ltd;
  •  Monica Cheesbrough (2002): District Laboratory Practice in Tropical Countries Part 1 & 2, Cambridge University Press; Cambridge, England.

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