Simple staining

Aim

To study the morphological shapes, size and arrangement of bacterial cells

Principle

In simple staining the bacterial smear is stained with a single reagent.  The simple stain can be used as a quick and easy way to determine cell shape, size and arrangements of bacteria.   Most bacteria have a defined shape that falls into the following morphological categories. Spherical or cocci, straight rod or bacilli, spiral or spirochetes. A few bacteria are pleomorphic and can change their shape.  For example Arthrobacter. 

Simple staining is performed by using basic stains.  Basic stains with positively charged chromogen is preferred because bacterial nucleic acid and certain cell wall materials carrying negative charge strongly attracts and binds to the cationic chromogen.  The most commonly used basic stains are methylene blue, safranin and crystal violet. 

Materials required

1. 24 hour old bacterial culture - Bacillus and Staphylococcus species

2. Crystal violet/Safranin

3. Equipments - bunsen burner, inoculating needle, staining tray, microscope

Procedure

1.  The bacterial smears were prepared and heat fixed in a clean glass slide

2. The smear was flooded in crystal violet or safranin for 1 minute

3. Crystal violet/Safranin was washed off gently using slow running tap water

4. The smear was air dried and observed under oil immersion objective

Observation

Purple coloured cocci in cluster were observed on staining with Crystal violet. 

Red coloured rods were observed on staining with Safranin.

 

Result

The given sample contain purple coloured cocci in clusters and red coloured rods against colourless background 

 

 

 

 

 

Preparation of Agar Slant and Agar Deep culture tubes

Aim

To prepare nutrient agar slant and deep culture tubes

Principle

Autoclaved agar medium poured into culture tubes and placed in slanting position results in slant after solidification. If the culture tubes are kept erect on solidification, it gives deep tubes.

Materials Required

Nutrient agar medium, sterilised test tubes, test tube stand, cotton plug, Bunsen burner

Procedure

1. 8-10 ml nutrient agar medium is poured into each test tube and cotton plugs put.

1. Tubes were transferred to test tube stand and autoclaved at 121^oC at 15 lbs pressure for 20 minutes.

2. One set of test tubes were kept in slanting position and second set of test tubes kept in test tube stand.

3. Medium was allowed to solidify.

Result

Agar slant and agar deep culture tubes were prepared.

Preparation of Nutrient Broth

Aim

To prepare nutrient broth for cultivation of bacteria.

Materials Required

Beef extract, Yeast extract, peptone, sodium chloride, agar agar, distilled water, conical flask, weighing machine, measuring cylinder, heater.

Procedure

1. Beef extract, yeast extract, peptone and sodium chloride were weighed to the required amount and added to a known volume of water.

2. They were mixed well and dissolved.

3. pH was adjusted to 6.8-7.2.

4. The medium was poured to conical flask or tubes in the required amount.

5. Those were plugged with cotton and autoclaved at 121^oC at 15 lbs pressure for 20 minutes.

6. The medium was then cooled to hand bearable temperature and poured into sterile test tubes as per requirement.

Result

A nutrient broth medium was prepared.

 

Preparation of Nutrient Agar Plate

Aim

To prepare nutrient agar plates for cultivation of bacteria.

Principle

Nutrient Agar is a basal solid medium which support the growth of a variety of common heterotrophic bacteria. It is one of the general purpose media. It is used for the isolation of mciroorganisms and to do some sensitivity tests. The pH of the medium is 6.8-7.2. The neutral range of pH range aids the growth of a wide variety of microorganisms. The nutrient agar media consists of:

Peptones- They are protein hydrolysate prepared by partial proteolytic digestion of meat, casein, soya meal, gelatin and other protein sources. They serve as source of carbon, energy and nitrogen.

Beef Extract- They are prepared by extracting water soluble components of beef tissues. It is a complex mixture of proteins, carbohydrates, lipids and other biochemical constituents.

Yeast Extract- An aqueous extract of yeast cells containing vitamins and other growth factors.

Agar agar- A complex polysaccharide extracted from the algae Gellidium. It is a solidifying agent with gelling temperature of 45^oC and melting temperature of 95^oC. It is not a nutrient source and is resistant to enzymatic dehydration.

 Materials Required

Beef extract, Yeast extract, peptone, sodium chloride, agar agar, distilled water, conical flask, weighing machine, measuring cylinder, heater.

 Procedure

1. Beef extract, yeast extract, peptone and sodium chloride were weighed to the required amount and added to a known volume of water.

2. They were mixed well and dissolved.

3. pH was adjusted to 6.8-7.2.

4. The agar was weighed and added to the medium and dissolved by boiling.

5. After the agar was dissolved properly, the medium was poured to conical flask or tubes in the required amount.

6. These were plugged with cotton and autoclaved at 121^oC at 15 lbs pressure for 20 minutes.

7. The medium was then cooled to hand bearable temperature and poured into sterile petri plates.

 Result

A nutrient agar plate was prepared.

 

 

 

 

Water-borne Diseases - Bacterial

Main Bacterial Pathogens

—  Vibrio cholerae

—  Escherichia coli

—  Salmonella typhi

—  Shigella flexneri

—  Campylobacter pylori

—  Legionella pneumophila

—  Leptospira

Diarrheal Diseases

Enteric fever/Gasteroenteritis/typhoid fever- Salmonella typhi

·         Characterized by fever and inflammation of intestine, ulcer formation, and other abdominal symptoms

·         Nonspecific symptoms - chills, sweats, headache, anorexia, weakness, sore throat, cough

·         The incubation period - 5 to 21 days.

·         Patients recover after 2-3 days; disease is limited to the GI tract in most cases

·         Organisms are excreted in urine and faeces in large numbers during the active phase of the disease

·         Max survival time 7 days; in natural waters above 15⁰C

·         Lack of adequate sanitation, major cause of the disease

·         Controlled by maintaining proper sanitation

Shigellosis (Bacillary dysentery)

·         Intestinal disease; most common among young children

·         Shigella species; S. dysenteriae, S. flexneri, S. boydii, and S. sonnei

·         S. sonnei (developed countries) S. flexneri (developing countries)

·         S. dysenteriae (most severe infections), S. boydii - not commonly isolated.

·         The organisms survive stomach acidity, pass through the small intestine, and adhere to the mucosal epithelium of the terminal ileum and colon

·         Destruction of the superficial mucosal layer - mucosal ulceration

·         Classic bacillary dysentery - characterized by diarrhea with blood, mucus, and painful abdominal cramping. Abundant neutrophils, erythrocytes, and mucus are found in the stool

·        An exotoxin (Shiga toxin) with enterotoxic and cytotoxic properties - isolated from S. dysenteriae; implicated in the development of intestinal lesions

·         Shigella species - remain viable in food and water for as long as 6 months

·         readily killed by chlorination of water, heat, and chemical agents

·         Infection - generally self-limited, spread through drinking water contaminated with human faeces

·         Shigella species carried primarily by humans and are not disseminated in nature

·     Most infections are passed by the faecal- oral route - Organisms excreted in faeces and urine; rarely survive more than 10 days outside

Cholera

·         Vibrio cholerae - ingested in contaminated food or water - multiply to large numbers on the mucosal surface and produce Cholera enterotoxin

·         The toxin causes hyper secretion of fluids and electrolytes into the intestinal lumen

·         After several hours to 3-days incubation, patients experience a sudden onset of severe watery diarrhea with vomiting and abdominal pain

·         The stool specimens are colorless and odorless, with mucuos flecks (rice-water stool)

·         Untreated, death from severe dehydration and loss of minerals

·         Large numbers of pathogens are excreted during the course of the disease - remain viable in natural waters till 7 days

·         Chlorination of water and standard disinfectants are able to destroy the organism easily

Campylobacter jejuni

·         Also causes diarrhea; transmitted through faeco-oral route

·      Campylobacter jejuni infection is one of the most commonly identified bacterial causes of acute gastroenteritis worldwide.

·         In developing countries, it is an important cause of childhood morbidity caused by diarrheal illness

·         among the most common causes of diarrhea in travelers from developed nations

·       Local complications of Campylobacter infections include cholecystitis (gall bladder infection), pancreatitis, peritonitis, and gastrointestinal hemorrhage

·   Extra intestinal manifestations of Campylobacter infection are quite rare and may include meningitis, endocarditis, arthritis, osteomyelitis, and neonatal sepsis.

·       Serious systemic illness caused by Campylobacter infection rarely occurs but can lead to sepsis and death. 

Other bacterial diseases - Non-Diarrheal

Legionnaires’ Disease (legionellosis)

·         Legionella pneumophila

·         Severe pneumonia; considerable morbidity, leading to death unless therapy is initiated promptly

·         commonly present in small numbers in natural bodies of water, such as lakes and streams

·         Multiply profusely in artificial  aquatic habitats like air conditioning, cooling towers and in water systems (e.g., showers, hot tubs)

·         Survive in moist environments for a long time, at relatively high temperatures, and in the presence of disinfectants such as chlorine

·         can parasitize amoebae in the water and replicate in this protected environment

·         Respiratory tract disease  in susceptible people who inhale infectious aerosols

·         Incubation period of 2 to 10 days;  fever, and chills, a dry non-productive cough,  headache

·         Can develop into multi-organ disease involving the gastrointestinal tract, central nervous system, liver, and kidneys

·         Maintain free residual chlorine in cold water supplies

·         Control the temperature of water (cold water below 20⁰C and hot water above 50⁰C)

Leptospirosis

·       Leptospira species; inhabit the kidneys of pet dogs and domestic livestock, excreted into natural waters in urine

·         enter through the abraded skin, oral or nasal mucosa, or conjunctiva following contact with urine-contaminated water by drinking, immersion of hands, or swimming  and infected wild life and domestic animals especially rodents

·         Leptospira icterhaemorrhagiae - associated with human disease; Weil’s disease

·         Fever, chills, headache, GI disturbance, and conjunctival redness

·         In 2 – 7 days, the patient becomes afebrile

·         After 1 – 3 day, a recurrence of fever, which persists for only couple of days

·         Jaundice, rash, hepatosplenomegaly, meningitis, and/or renal damage

Ethanol Production by Microbial Fermentation

 Ethanol and concentrated alcoholic drinks by fermentation- used since long for consumption.

 Microbial Fermentation in early days. But now, chemical synthesis

 In 1906 when the Industrial Alcohol Act was passed, the production of industrial alcohol, ethanol, became commercially feasible on a large scale.

 There are various uses of alcohol such as, 

1. Ethanol is used as an organic solvent in many different products as paints, perfumes, markers inks, varnishes, soaps, and other products like explosives.

2. The most popular use of ethanol is as motor fuel and fuel additive (gasohol)- renewable source for environmentally friendly fuels.

4. Ethanol is used as a potent food preservative in homes.

5. Ethanol is used in making antiseptic soap and cosmetics as it is effective against bacteria, fungi, and many viruses, but is not much effective against bacterial spores.

6. Ethanol is routinely consumed in different forms around the world like beer, wine, gin, whisky, etc. Beer is the most popular ethanol beverage in the world.

7. Ethanol plays an important role in making drugs and pharmaceuticals.

8. Ethanol is used in the preparation of essences and flavorings

9. Ethanol is used as the fluid in thermometers.

10. Ethanol is used in preserving biological specimens.

 Microbial Fermentation 

• Ethanol fermentation is a biological process in which sugars are utilized to produce ethanol and carbon dioxide as metabolic waste products.
• This process is carried out by yeast cells that converts glucose to CO₂ and alcohol.
• C₆H₁₂O₆ (glucose) → 2 C₂H₅OH (ethanol) + 2 CO₂ (carbon dioxide)
• Before fermentation, one glucose molecule is converted into two pyruvate molecules by glycolysis. 

• Under aerobic condition, some species of yeast (e.g., Kluyveromyces spp.) will oxidize pyruvate completely to carbon dioxide and water which is termed as cellular respiration.
• These species of yeast will produce ethanol only under anaerobic condition.
• So ethanol fermentation is classified as anaerobic process.
• However, many yeasts such as Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast) will produce ethanol even under aerobic conditions
  
  
• Microorganisms

Many yeasts and bacteria such as Zymomonas mobilis are suitable for ethanol production. But, all yeasts are not suitable for brewing. Choice of yeast for the alcohol production depends upon the composition of the medium, particularly the type of carbohydrate used in the medium.

.

Raw materials	Fermentation Strain
Starch and sugar	Saccharomyces cerevisiae
Lactose of whey	Candida pseudotropicalis
Sulfur waste liquor	Candida utilis

 Selected strain should 

ü  grow rapidly.

ü  tolerate high concentrations of sugar.

ü  be able to produce abundant amounts of alcohol

ü  be resistant to the produced high concentrations of alcohol. 

Ethanol Fermentation

 Three stages

1. Media Preparation
2. Fermentation
3. Product harvesting/Distillation

                                   (contd)

References

Principles of Fermentation Technology: ( Peter F. Stanbury, Allan Whitaker )

Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications)

A Text Book of Industrial Microbiology: (2nd edition By Wulf Crueger & Anneliese Crueger)