Staining Techniques to Observe Stained Cells

Microorganisms are invisible to the naked eye. Therefore for the purpose of studying, they are cultured. Staining will kill microorganisms. Dyes are used to staining and smears are prepared. Most commercial dyes are salts in which one of the ions are colored. Eg: Methylene blue (MBCl).

MBCl   =   MB⁺ + Cl^-

There are two kinds of dyes. Basic dyes and acidic dyes. Methylene blue, crystal violet, carbon fuchsin are basic dyes in which the positive ion gives the color. Basic dyes will stain the acidic components of the cell such as the nucleus.

Salts in which the negative ion gives the color are called acidic dyes, stains basic components of the cell such as cytoplasm and granules. Eg:  Safranin, Congored, and Acid Fuchsin.

Methods of Staining

• Simple staining

A single dye application.Mainly basic dyes such as Methylene blue, crystal violet, carbolfuchsin used.

• Differential staining

Use more than one dye. This method distinguishes one type of cell from another.

Eg: Gram stain, KOH procedure (gram reaction).

Negative Staining and Hanging Drop Procedure

Observation of living cells follows below procedures.

Negative Staining

Microorganism will not stain in this method, but the medium/environment will stain. Staining will kill the microbe hence negative staining provides us the facility to observe the live cells. This method will be discussed more under the topic practical.

 Medium is stained but not cells

Hanging Drop Procedure

In this technique, a drop of medium containing cells is allowed to hang in the cavity slide. This method is used to study the motility of microorganism. There are two movements that can be observed, true movement and Brownian movement. Microorganism changes its position in a true movement whereas in Brownian movement the organism oscillates at its place and does not change the position in the field. Brownian movement can be explained based on the bombardment of small particles suspended in the fluid, with the cell. This method will be discussed more under the topic practical. 

Most Probable Number (MPN) Test for Water (Viable Cells)

Theory

The routine microbial examination of water to determine its portability is not based on the isolation and identification of pathogenic microorganisms but based on finding a microorganism whose presence indicates that water has been contaminated with an intestinal origin, and therefore the possibility of the presence of pathogenic microorganisms.

The fecal contamination of drinking water is evaluated by using coliform bacteria as an indicator. An indicator organism is an organism that can be used as an indication of pollutions.

Coliforms ferment lactose within 48 hours and produce lactic acid and carbon dioxide.

Lactose -------->  Lactic acid  + carbon dioxide

In the coliform test, determining the sanitary quality of water involves 4 stages such as;

• Presumptive test
• Confirmed test
• Completed test
• Positive completed test

In this lab presumptive test is carried out. In the presumptive test, the indicator is phenol red. The color changes from red to yellow. In the basic medium, phenol red is red whereas in the acidic medium it is yellow.

Procedure

First two serial dilutions 10^-1, 10^-2 of water samples provided, were prepared. Next 1ml aliquots were transferred (each from 10⁰, 10^-1, 10^-2 tubes) into the lactose broth in Durham fermentation tubes. This was done in duplicates. Finally, the tubes were incubated at 35⁰C for 48 hours.

Observation

Sample	No of positive tubes						CFU/ml	CFU/100ml
	100		10-1		10-2
	1	2	1	2	1	2
Tap water	0	0	0	0	0	0	0	0
Tubewell water	0	1	0	1	0	1	2	2*100 = 200
Rain water	1	1	1	1	0	1	70	70*100 =7000
Bottled water	0	0	0	0	0	0	0	0
Well water	1	1	1	1	0	1	70	70*100 =7000
River water	1	1	1	1	1	0	70	70*100 =7000
Drain water	1	1	1	1	1	1	100	100*100 =10000
Stream water	1	1	1	1	1	1	100	100*100 =10000

Discussion

The presence of specific indicator organism signals that a given water sample is contaminated with pathogens. The most widely used indicator for microbial water contamination is the coliform group. Coliforms are used as indicators of water contaminations because many of them inhibit the intestine of humans and other animals in large numbers. Thus their presence in water indicates fecal contamination. Therefore most probable number method can be used to detect and enumerate coliforms in water.

Lactose fermentation after 48 hours at 35⁰C indicates a positive presumptive test for the presence of coliforms in the sample. In this practical, the indicator is phenol red. In a neutral medium, phenol red gives an orange color while it gives a yellow color in acidic medium. Hence the color changes from orange to yellow shows that the medium has changed from neutral to acidic. That indicates the formation of lactic acid due to lactose fermentation. The gas bubble in the fermentation tube also indicates the lactose fermentation. Gas bubble is carbon dioxide. By referring to a statistical table no of coliforms per 100ml can be obtained (MPN Table).

According to the above results, the highest number of colony forming unit (CFU)/100ml was shown by drain water and stream water. Stream water flows through various areas and also polluted water from industries which are coming from drainages used to flow into streams. Hence both drain and stream water are highly polluted. Thus the coliform contaminations may be higher, due to the exposure to various situations. Next highest is well water, river water, and rainwater. Well water does not flow. River water flows but does not expose to various areas as the stream water. This is why CFU of river water does not exceed the CFU of stream water. Although rainwater is exposed it does not contaminate with pollutants. But CFU value of rainwater has a high value. It may be due to instrumental or experimental errors. This can occur due to unclean containers or when counting a deformed gas bubble may be counted. Because when the gas bubble is very large the bottom of the gas bubble is difficult to identify. Both tap and bottled water have zero values, which indicates these two samples are not contaminated with coliforms. Since tap water is purified before distributing to houses it is already checked for coliforms. Only if it is free from coliforms the distribution is done. Bottled water is also purified and sterilized.

For the success of the results, it is important to use sterile containers to collect water samples. And a wide-mouthed, sterile, glass-stoppered bottles can be used. When collecting tap water, let the water to flow initially and then take the sample, whereas, in a stream, water is collected below the water surface. If transportation is a necessary water sample is packed in ice. Otherwise, due to environmental conditions, the activity of the organism may change. To inhibit further activities of coliforms, after 48 hours the samples should place in a refrigerator.

According to the national water and drainage board, total coliforms equal to 10 while fecal coliforms equal to 0, are acceptable values in drinking water /100ml. These values can vary depending on the country.

IMViC Test

Theory:

This is a group of tests used particularly in the identification of the family Enterobacteriaceae. This group consists of Escherichia coli, Salmonella sp, Shigella sp, Enterobacter sp, Proteus sp, Eruinia sp. 

IMViC test contains several tests such as;

• I = Indole test
• M = Methyl red test
• V = Voges Proskauer test
• C = Citrate utilization test

Indole test carried out in sim agar media;

Methyl red test carried out in MR-VP Broth;

Reaction with E.coli;

E.coli can change the pH of the medium into an amount of 4.

Reaction with E.aerogenes;

E.aerogenes can change the pH of the medium into an amount of 6.5.

Voges Proskauer test carried out in MR-VP Broth;

This changes the pH around 6.0.

Citrate test carried out in Simmons citrate medium;

Color change in Indole test is from pale yellow to red and methyl red test pale yellow to red. In Voges Proskauer test is from yellow to red and in citrate test, it is from green to blue.

Procedure:

Indole Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with tryptone broth in separate test tubes. Next, these were incubated at 37⁰C for 2-5 days. Finally, the indole was tested by Kovac’s method.

Kovac’s reagent was added carefully to the test medium dropwise to form a layer at the top of the medium. Finally, the tube was gently agitated with a rotary motion.

Kovac’s reagent: 5g of para dimethylamino benzaldehyde dissolved in a mixture of 75ml of amyl alcohol and 25ml of concentrated HCl.

Methyl red Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with MR-VP (dextrose phosphate medium) in separate test tubes. And this was incubated at 37⁰C for 48 hours. Finally, a few drops of an alcoholic solution of methyl red were added to the tube.

Voges Proskauer Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with MR-VP (dextrose phosphate medium) in separate test tubes. And this was incubated at 37⁰C for 2-5 days. In order to test Acetylmethylcarbinol, 0.5ml of naphthol solution (5% alcoholic) and 0.5ml of 40% KOH containing 0.3% creatine were added to about 3ml of the test medium. Next, the tubes were stoppered and shaken vigorously and were placed in a slanted position for 5-15 minutes. 

Citrate Utilization Test

First, the colonies of E.coli and E.aerogenes from the completed test were used to inoculate with Koser’s citrate medium. And Bromothymol blue was added as an indicator.

Observations:

	Test	Observation	Conclusion
	Indole Test	A red ring was formed on top of the peptone solution which was incubated with E.coli and the pale yellowish color of the solution remained the same. No such formation of a red ring has occurred in the broth which was incubated with E.aerogenes.	E.coli present.
	Methyl red Test	MR-VP medium with E.coli was formed a red ring on top of the solution and color of the solution remained the same. MR-VP medium with E.aerogenes has not formed a red ring.	E.coli present.
	Voges Proskauer Test	MR-VP medium with E.aerogenes turned the yellow solution into dark red whereas no specific change in MR-VP medium with E.coli.	E.aerogenes present.
Citrate Utilization Test		Koser’s citrate medium with E.aerogenes turned the green solution into blue whereas no change in color in Koser’s citrate medium with E.coli	E.aerogenes present.

Discussion:

In order to provide definite biochemical proof that the isolated organism is indeed E.coli a series of test, usually referred to as the IMViC tests are used. These can be used both to characterize E.coli and to differentiate it from Enterobacter aerogenes. Colonies from the completed test used to inoculate several different media. And this test can be used to identify other species in family Enterobacteriaceae.

A peptone medium rich in the amino acid tryptophan is inoculated and allowed to grow for 24 hours. E.coli makes the enzyme tryptophanase which forms indole, pyruvic acid, and ammonia from tryptophan. Since E.aerogenes cannot catabolize tryptophan, testing for the presence of indole differentiate these two organisms. Formation of red ring indicates the presence of E.coli.

P-dimethyl aminobenz-aldehyde     +    Indole ------> Cherry-red compound

Methyl red is an acid-base indicator that turns red in a slightly acidic medium. Thus if methyl red is added to a culture medium containing glucose in which an organism has grown for 18 to 24 hours, a red color will be observed which would indicate that organic acids had been formed as a result of fermentation of the glucose. E.coli forms large amounts of acids, which make methyl red positive. E.aerogenes on the other hand, carries out a 2,3-butylene glycol type of fermentation and thus produces only small amounts of organic acids which makes methyl red negative.

The Voges Proskauer test detects the presence of acetoin (Acetylmethylcarbinol), the immediate precursor of 2, 3-butylene glycol fermentation which is positive for E.aerogenes and negative for E.coli and the positive result is indicated by a pink ring.

Acetyl methyl carbonyl  +  Napthol ------>  Diacetyl +Guanidine (group of  peptone)

This reaction is carried out in the 40% KOH oxidation. The pink color is obtained both by Diacetyl and guanidine.

Citrate test merely determines whether the organism in question can grow using citrate as its sole source of carbon. Although both E.coli and E.aerogenes possess the necessary enzymes to metabolize citrate, only E.aerogenes can use it as a carbon source. E.coli cannot use citrate because citrate cannot enter the cells of E.coli. This test, therefore, determines the difference in the ability of these two organisms to transport citrate across their cell membranes.

                       Citrase

Citric acid -------> Oxalic acid + acetic acid + pyruvic acid + CO₂ excess

2CO₂ +  2 Na⁺  + 2 H₂O ----> Na₂CO₃ ----> Colour change from green to blue

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