Curd Microorganisms: Gram stain introduction, Procedure and Result-Inter

Curd Microorganisms: Gram stain introduction, Procedure and Result-Interpretation

Introduction of Curd Microorganisms

Curd microorganisms in gram stain are bacteria, Lactobacillus, and fungus, yeast cells as shown above picture. Curd is a dairy product and it is obtained by coagulating milk in a process called curdling. The coagulation can be achieved artificially by adding rennet or any edible acidic substance such as lemon juice or vinegar. The increased acidity causes the milk proteins (casein) to tangle into solid masses or curds. Whereas, raw milk alone or pasteurized milk which is then added with lactic acid bacteria will also naturally produce curds, and sour milk cheeses are produced this way.

Preparation of curd requires bacteria are-

Lactobacillus delbrueckii subsp. bulgaricus
and Streptococcus thermophilus

The main ingredient in curd is casein. It is the reserve protein of milk that gives the product its white main color. The primary biological value of the curd is in its high protein content i.e. usually 10-12% which varies a little by curd variety.

Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria and is useful for fungi. Yeast cells are Gram-positive. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.

Principle

The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. The alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet.

Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.

Requirements for Gram stain of Curd microorganisms

a) Compound light microscope

b) Reagents and glasswares

Bunsen flame
Wire loop
Clean grease-free slides
Marker pen
Crystal violet (Basic dye)
Gram’s iodine(mordant)
95% ethanol (decolorizing agent)
1% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen ( curd)

Preparation of Smear for Curd Microorganisms

Take a clean, and grease-free slide for making a smear.
Take one or two loopful of watery curd and place them on the slide with a bacteriological loop. ( If the curd is solid or semi-solid, take a loopful of 0.85% saline i. e. physiological saline, and place it on the center of the slide. With a straight wire touch the curd and emulsify in the saline drop forming a thin film.)
Then with a circular movement of the loop, spread the cell suspension into a thin area.
Allow the smear to air dry.
Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.

Procedure

Cover the smear with crystal violet and allow it to stand for one minute.
Rinse the smear gently under tap water.
Cover the smear with Gram’s iodine and allow it to stand for one minute.
Rinse smear again gently under tap water.
Decolorize the smear with 95% alcohol.
Rinse the smear again gently under tap water.
Cover the smear again gently with safranin for one minute.
Rinse the smear again gently under tap water and air dry it.
Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Observation

Positive Control: violet color, round in shape in single, pairs and cluster

Test: Purple color, rod, and oval-shaped organisms

Negative Control: red in color and rod in shape

Result and Interpretation

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-positive bacilli ( ? Lactobacillus ) and Yeast cells ( fungi)

Negative Control(NC): Gram-negative bacilli as shown above image.

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