Microbiology Practical

Practical 3 :  Staining

3a.Simple staining :

 Monochrome staining

Aim : To observe the morphology i.e. shape, size & arrangement of bacteria in a given bacterial suspension by monochrome staining.

Theory : Bacterial cell morphology may be examined by observing living unstained organisms or dead cells stained with stain the idea of staining microorganisms was introduced in microbiology by R. Koch in 1881. Living bacteria are almost colourless and therefore don't posses sufficient contrast in which they are suspended staining microorganisms make them contrast in colour with surrounding so that they can be radially visible.

Principle : 'A salt is composed of positively charged & negatively charged ions. Stains are the salts in which one of the ion is coloured'. E.g. methylene blue actually methylene blue chloride dissociate as methylene blue chloride ➝ methylene blue ㊉ + chloride ㊀.

The colour of it is  positively charged methylene blue ion. Hence the stain is basic stain. Bacteria have slightly negative charged at the surface, when the pH of surrounding is at neutrality. Thus, with charged bacteria cell combines with positively charged methylene blue. The result is cell get stained.

Bacterial cell ㊀ + methylene blue ㊉ = coloured stained bacterial cell

 Thus, it is the difference in the charge that produce the affinity between cell & stain.

The stains are either acidic or basic. If colour of stain is positively charged ion, it is a basic stain.

E.g. Crystal violet, Basic fuschin, Saffranin, MB etc.

If colour of stain is negatively it is acidic stain.

E.g. Nigrosine, congored, India ink, Eosin, etc.

  In monochrome staining, fixed smear is flooded with a single solution of basic fuschin for specific period of time after which the stained solution is washed with water.

Requirement :

1) Clean grease free slide.

2) Inoculating needle.

3) Given bacterial suspension.

4) Basic stain

 E.g. 1% crystal violet solution

Composition of the cell: 

a) crystal violet powder : 1gm

b) Distilled water : 80ml

c) Ethanol : 20ml

d) Ammonium oxalate : 0.89gm - (800 mg)

Procedure : 

Part 'A' : Preparation of slide

A smear was prepared on clean grease free slide aseptically. By placing a drop of suspension with the help of inoculating needle and spreading to form thin even smear. 

The smear was allowed to dry in air.

Heat fix the smear by passing the slide 3 times through flame with the film slide up.

Part 'B' : Staining of microorganisms

The slide was placed with fixed smear on a staining rack over to absin.

Each of the smear was flooded with steam & allowed to react for 2 min.

The slide was washed with tap water.

A drop of cedar wood oil was placed on dried smear & observed under oil immersion objective (100X) of compound microscope & recorded the size, shape & arrangement of coloured bacteria.

Result of monochrome staining :

In the given bacterial suspension, cocci & rod shaped bacteria are observed by monochrome staining.

Observation (monochrome staining)

Negative staining / Indirect staining method :

Aim : To observe the shape, arrangement, structure by negative or indirect staining.

Theory : In negative staining the bacterial cells are not stained and the cell remains colourless but the background is stained. So, it is known as relief staining because background get stained, while bacteria stained out in relief has constant area. This method has an advantage over direct staining method. Negative staining gives more accurate natural views of bacteria. It is likely to get a clear picture of its size & shape.

Principle : "Nigrosine eosin (sodium eosinate), India ink, congored are some of the acidic stains. The colouring property of acidic stain is negatively charged ions. So, it will not readily combine with another negative ion. Thus, acidin dye does not stained. The negatively charged bacterial cell instead it forms a deposit around the cell & stain the background.

Bacterial cell ㊀ + Nigrosine ㊉  ➝ No colouration bacterial cell but background stained.

Requirement :

- Clean grease free slide.

- Inoculating needle 

- Bacterial suspension

- 10 % nigrosine

Composition - Nigrosine - 10gms 

                   Distilled water - 100ml

Procedure :

Part 'A' 

10 gms of nigrosine crystals are dissolved in small amount of warm distilled water & then finally volume made upto 100 ml. Aseptically transfer a loopful of bacterial suspension at the edge of clan slide. Add a drop of nigrosine solution to the drop of suspension.

Mix the suspension & stain with the help of needle spread the drop in the form of thin film with the edge of another glass slide.

Part 'B' 

Air dry the film.

Put cedar wood oil on it & examine under 100X oil immersion objective of compound microscope.

Result :

In the given bacterial suspension colourless bacilli are observed against bluish black background by negative staining method.

Observation (Negative staining)

3.b Differential Staining : Gram staining 

Aim : To stain and there by to difference the type of microorganisms in given bacterial suspension by gram staining method.

Theory : Gram staining is most careful staining procedure . This is a type of differential staining method because by using the staining it is possible to differentiate bacteria into two groups positive & gram negative bacteria.

The grams staining method require 4 different staining solutions -

1) Basic stain 

2) Mordant 

3) Decolouring agent 

4) Counter stain.

A mordant is a substance which increases the affinity between bacterial cell & stain. Under the action of mordant a bacterial cell & stain strongly reacts & get stained. In gram's staining method, grams iodine is used as a mordant.

Iodine is an oxidizing agent. It oxidises a component which are more acidic & have greater affinity for  basic stain. 1% crystal stain solution is used as a primary basic stain. 95% ethanol is used as decolourising agent. Saffranin or basic fuschin 0.5% used as counter stain.

Some stained cells have tendency to decolourize much more easily than other . In gram's staining this variation in rate of decolourisation is used to differentiate different types of bacteria in given bacterial suspension. The counter stain, stain decolourised cells. It is also a basic stain but of different counter than primary basic stain crystal violet even after decolourisation are called gram positive bacteria.

Requirements :

1) Clean grease free slide.

2) Inoculating needle 

3) Given bacterial suspension 

4) 1% crystal violet 

Composition : - crystal violet powder : 1gm

    Distilled water : 80ml

    Ethanol : 20ml

    Ammonium oxalate : 0.8gm

5) Gram's violet composition : - Iodine crystal : 1gm

Potassium iodine : 2gm

Distilled water : 300ml 

6) 95% ethanol

7) 0.5% saffranin / Basic fuschin 

composition :- saffranin - 0.5gm 

Distilled water - 100ml

Procedure :

1) Prepare a thin smear on clean grease free slide with the help of inoculating needle aseptically.

2) Air dry & heat fix the smear.

3) Flood the smear with 1% crystal violet solution for 2 min.

4) discard the crystal violet & flood with gram iodine solution for 1 min.

5) remove iodine solution, decolourize the smear with 95% ethanol, Allow to react for 20 -30 sec.

6) Wash the slide with counter stain i.e. 0.5 saffranine / basic fuschin 2 - 3 min.

7) Wash the slide with tap water dried the smear & observe under oil immersion objective.

Result :

In the given bacterial suspension, violet coloured gram positive, rod shaped isolated bacteria & red coloured gram negative short rods are observed in grams staining method.

Observation (Gram's staining)

3c. Structural staining 

 (i) cell wall :- A) Ringen -et al method

Aim : To stain the bacterial cell wall by ringen et al.

Theory : Beneath the capsule & surrounding the cytoplasm is the cell wall. It is rigid structure which gives definite shape & size to bacterial cell. It is composed of peptidoglycan. Because of its thickness & chemical nature. It is resistant to staining. It is normally difficult to separately stain the cell wall from the cell.

Principle : "Cell wall is made positively charged by treatment with cationic surface agent. e.g. Tannic acid which is a mordant. Thus, it can now take up stain, cytoplasm remains negative charge and can be stained by basic dye. In Ringen et al method , crystal violet & congored are used. Crystal violet acts as a stain & congored as a decoloriser which decolourises the cytoplasm.

Requirement :

1) Clean grease free slide

2) Inoculating needle 

3) Given bacterial suspension

4) Tannic acid - 5% solution

Comp :- Tannic acid powder - 5gms

         Distilled water - 100ml

5) Crystal violet solution (0.5%)

composition : Crystal violet powder - 0.5gm

                      Distilled water - 100ml

6) Congored (0.5% solution) 

composition : Congored powder - 0.5gm

                      Distilled water - 100ml

Procedure :

A thin even smear is prepared on clean grease free slide.

Air dry it. Do not heat fix.

Tannic acid was poured on smear & allowed it to react for 30 to 60 min. The slide was then washed with water gently. The crystal violet solution was poured on the smear. Allows it to react for 2 min. Discarded & gently washed.

A thin film of 0.5% congored was prepared on the smear. It was dried & observed under oil immersion objective of the compound microscope.

Result :

In the given bacterial suspension, on red background , bacteria with colourless cytoplasm & violet cell wall are observed by Ringen et al method of cell wall staining.

Observation [cell wall (Ringen et - al method)] 

Chance's method 

Aim : To stain bacterial cell wall by chance's method.

Principle : "Bacterial cell is stained pink with new fuschin and then only cytoplasm is decolourised with congored solution & cell wall remains pink".

Requirement :

1) Clean grease free slide.

2) Inoculating needle.

3) Given bacterial suspension

4) New fuschin solution

Composition : Basic fuschin - 0.5gm

                       Distilled water - 100ml

5) Congored solutions : 0.5%

Procedure :

A thin even smear was prepared on clean grease free slide.

Air dry do not heat fix.

Flooded the smear with new fuschin solution & allowed to react for 3-4 min New fuschin solution is discarded & washed.

Prepared a thin film of congored solution on dried smear the help of another slide, dry the film & observed under oil immersion objective of compound microscope.

Result : 

In the given bacterial suspension, red coloured bacterial cell wall with colourless cytoplasm are observed on red background by chance's method of cell wall staining.

Observation [Cell wall staining (Chance's mehod)]

3 (C) Structural staining 

(ii) PHB staining : Burdon's method 

Aim : To stain PHB granules by Burdon's method.

Theory : 

All the bacterial cells at various stages of their growth passes deposists of components which are believed to the their reserved food materials. These reserve food materials are primarily lipids. Lipid is one of the most important constituent of cell.

It is found in cell wall & cell membrane of cell. Lipids are also found as globules in cytoplasm, They are mainly composed of poly B Hydroxy butaric acid, PHB is polymer of C4 fatty acid, without glycerol.

Fat globules are also known as sudanophilic granules & found mainly in old cultures. Bacillus cereus produced large sudanophilic bodies even in simple cultural conditions. They are believed to be reserve source of carbon. Fat globules are not easily stained by water soluble dyes but can be demonstrated easily using fat soluble dye. Dye used in these technique is more soluble in the lipid than in solvent staining solution. So, an application of staining solution dye moves into lipid material of cell & is restained there.

Principle : 

Sudan Black B stain is used. This is a fat soluble stain which stains the lipid layer by getting dissolved in it, saffranin is the counter stain which stains the cytoplasm of bacterial cell.

Requirement : 

1) Clean grease free slide.

2) Inoculating needle.

3) Bacterial suspension (old culture)

4) Sudan Black B Stain 

Composition : 

Sudan Black B Powder - 0.3g

Ethanol (70%)               - 100ml

5) Xylene 

6) 0.5% Saffranin

Procedure : 

Prepare a thin even smear of bacterial suspension on clean grease free slide, air dry & heat fix.

Flood the smear with Sudan black B & allow to react for 10-15 min.

B Drain off the excess stain & dry it.

Do not wash with water.

Rinse the smear with xylene & blot dry.

Don't wash with water.

Counter stain with 0.5% saffranin for 5-10 sec.

Rinse the water, blot dry & examine under oil immersion objective.

Result :

Black PHB granules central in position in a red vegetative cell were observed by using Burdon's method.

Observation (PHB staining)

Practical 6 : Preparation of culture media 

i) Nutrient broth 

Aim : To prepare 100 ml of nutrient broth

Theory : The most common medium used for cultivation of bacteria is nutrient broth. the ingredients are peptone, beef extract, NaCl and water proteins is a simple nitrogen and carbon source utilized by microorganisms. Beef extract is the complex nutrient which contains organic compounds such as vitamins, proteins, minerals and salts. Thus it supplies nutrients for microbes. Sodium chloride i.e. NaCl maintains osmotic pressure of the medium. The pH of the broth is generally adjusted to 7.4 because mast of the bacteria grow best at neutral pH range. The liquid medium is known as broth.

Requirement :

1) Nutrient broth

Composition - 

Peptone - 1gm

Beef extract - 0.3gm

NaCl - 0.5gm

Distilled water - 100ml

pH - 7.4

For adjusting the pH of broth 0.1N HCl and 0.1 N NaOH is required.

2) Conical flask

3) Chemical balance 

4) pH meter/ pH paper

5) Glass rod 

6) Factional weight box

7) Butter paper

Procedure : 

The ingredients of the medium were weighed accurately with the help of chemical balance.

The chemical ingredients were when dissolved in distilled water in a conical flask.

The pH of the broth was adjusted to 7.4 with help pH paper and 0.1N HCl and 0.1 N NaOH.

The broth is then distributed in test tube of 5ml it was plugged with cotton, wrapped with paper.

All the test rube were kept for sterilization in autoclave at 121°C temp. at 15 lbs pressure for twenty (20) min.

Result :

100 ml of nutrient broth was prepared.

ii) MacConkeys agar

Aim : To prepare mac Conkey's agar

Theory : Mac Conkeys agar is selective as well as differtial medium. It is used for cultivation of entric bacteria. It contains the bile salt i.e. sodium taurocholate which inhibits the non intestinal bacteria, so it is called as "selective medium" as it sects the growth of coliform organisms over non coliform organisms.