ASSIGNMENT

FOOD MICROBIOLOGY

Submitted to- Dr. Himjyoti Dutta (Assistant Professor)

Dept of Food Technology

Submitted by: Lalthazuali Hnamte

Roll no: 23/CPSY/20

1st Semester
Classification and Identification of Microorganisms

As microscopic organisms, microorganisms, also referred to as microbes, are too small to be

seen with the human eye. There are two types of microorganisms: eukaryotic and prokaryotic.

An undeveloped cell type known as a prokaryotic cell is defined by its lack of a nucleus.

Moreover, prokaryotes lack cellular organelles that are membrane-bound. Prokaryotes are only

single-celled organisms. Eukaryotic cells are those that have membrane-bound organelles in

addition to a real nucleus. Eukaryotes can have several cells or only one. The major groups of

microorganisms are bacteria, fungi, algae, protozoa and viruses.

[Link] : Bacteria are unicellular prokaryotes and can appear in pairs, tetrads, chains or

clusters. Some have flagella for locomotion, they comes in different shapes and sizes eg: cocci,

vibrios,bacillus, spirila etc. Some have capsule- protective body which is typically a hydrated

complex that covers the outermost part of the cell all. Some of them form a spore: reproductive

endospore/protective exospores. Spores are the unicellular and reproductive structures, which are

greatly resistant to extreme environmental conditions, like high temperature, pH, salt

concentration, etc. Endospore is the common type of spores produced mainly by bacteria and are

formed inside the mother cell, whereas, exospores are formed towards the end of the mother cell

and released as bud. Some of the bacteria are gram positive and gram negative. The gram-

positive bacteria retain the crystal violet colour and stain purple whereas the gram-negative

bacteria lose crystal violet and stain red. Thus, the two types of bacteria are distinguished by

gram staining. The cell wall of gram-positive bacteria is composed of thick layers peptidoglycan.

The cell wall of gram-negative bacteria is composed of thin layers of peptidoglycan. Gram-

positive bacteria produce exotoxins. Gram-negative bacteria produce endotoxins. Identification

of bacteria species is done on the basis of -

• Morphology (microscopic observation, colony shape and height)

• Metabolism (colour of colony,colour changes in media)

• Cell components (compositional analysis, type of a component)

Genotypical traits:

• DNA profiling (fingerprinting)

• RNA

2. Fungi: Fungi are eukaryotic organisms that include molds and yeasts, among other microbes.

They store food as starch. Spores are used to reproduce them, and they can have one or more

[Link] may be viewed as being unicellular fungi in contrast to the moulds which are

multicellular. Yeasts can be differentiated from bacteria by their large cell size and their oval,

elongate, elliptical or spherical cell shapes. Typical yeasts cells range from 5-8um in diameter

with some being even larger. Older yeasts cultures tend to have smaller cells. Most of those of

importance in foods divide by budding or fission. Moulds are filamentous fungi that grow in the

form of tangled mass that spreads rapidly and may cover several inches of area in a very short

period. It is also referred to as mycelia growth. Mycelium is composed of branches of filaments

referred to as hyphae. The molds of great importance in foods multiply by ascorpores or conidia.
3 Algae: Eukaryotic, photosynthetic organisms make up algae cells. They may be multicellular

organisms or unicellular ones. They are typically found here, where the moisture content is

suitable. While certain algae can develop symbiotic relationships with other animals, algae are

free-living creatures. Because of their poorly defined bodies, they lack features like roots, stems,

and leaves.

4. Protozoa: Protozoa are heterotrophic, unicellular eukaryotic creatures. They don't have a cell

wall, thus they can live freely or as parasites. Because they don't have a hard cell wall, they can

be found in both freshwater and saltwater and can take on a variety of shapes. A few of them

infect humans and animals with different diseases. Certain species can be found in harsh

conditions, such as hot springs, while others develop cysts to withstand arid surroundings.

5. Viruses: Viruses are classified as non living organisms and are most simplest structure. They

are filterable, ultra microscopic particles and can be cultivated only on live tissues. Viruses

consists of a core of nucleic acid DNA or RNA and protein coat. It is commonly believed that

some of the viruses are responsible for food borne diseases in humans. Contaminated water and

food are important carries of hepatitis viruses. Food and mouth disease (FMD) causing virus in

cattle can be transmitted to human beings through foods.

Factors responsible for microbe grot h in foods

Microbial growth is an autocatalytic process, no growth will occur without the presence of at

least one variable cell and the rate of growth will increase with the amount of variable biomass

present. Grot h is the increase in cellular constituents that may result in increase in cell number

eg: hen microorganisms reproduce by budding or binary fission and may also result in increase

in cell size eg: coenocytic microorganisms have nuclear divisions that are not accompanied by

cell divisions.
6 basic factors important for the growth of micro-organisms:

1. Oxygen: A very important requirement for cellular growth and metabolism is oxygen.

Microbes can be grouped into five different categories based on their requirement for molecular

oxygen. Strict or obligate aerobes must have O2 for growth because O2 is used as the terminal

electron acceptor for oxidative phosphorylation. In contrast, strict or obligate anaerobes cannot

grow in the presence of O2 and may be killed by trace amounts of O2. Microaerophilic

organisms need a small amount of O2 for growth but too much O2 will kill them. A subset of

anaerobes can tolerate O2 but they do not use it. They are called aerotolerant anaerobes.

Facultative anaerobes grow best when O2 is available, but they can also grow, though not as

well, if O2 is not present.

2. Temperature of processing and storge of food: Temperature is one of the most important

environmental factors, controlling the rate of cell division of microorganisms. There are three

basic temperatures- minimal, optimal and maximal temperature.

3. Moisture (water activity): water activity is an important property that can be used to predict

food safety, stability and quality. The various applications of water activity includes maintaining

chemical stability of foods, minimizing non enzymatic browning reactions and spontaneous

autocatalytic lipid oxidation reactions, prolonging the desired activity of enzymes and vitamins

in foods, optimizing the physical property of foods such as texture

4. pH: The range of pH in which an organisms grow is defined by three cardinal points: the

minimum pH below which the organisms grow the maximum pH above which the organism

cannot grow and the optimum pH at which the organisms grows the best. Microorganisms
which grow at an optimum pH well below neutrality (7.0)are called acidophiles. Those which

grow best at neutral pH range are called neutrophiles and those that grow best at alkaline

conditions are called alkalophiles. In general, bacteria grow faster in the pH range of 6.0-8.0,

yeasts 4.5-6.5, fungi 3.5-6.8.

5. Formations of metabolites during growth:

• Metabolism- all the reactions in our body that helps in maintaining the grot h and

reproduction of living cells.

• Primary metabolites- they are small molecules of living cells, intermediates or end

products of the pathway related to synthesis of microbial cells in the growth phase

include alcohols, amino acids, nucleotides, organic acids, polyols, vitamins, and

enzymes.

• Secondary metabolites- accumulate following active growth, have no direct relationship

to synthesis of cell material and natural growth include antibiotics and toxins.

6. Preservatives and sterilizing agents: this process is called thermal processing that is done by

killing the microorganisms they preserved the food in order to make them stay longer.

Preparation of media:

1. Composition: peptone, yeast/beef extracts, solidifying agent, salt concentration for cytoplasm,

distilled water, clean glass are, micropipette, cotton are the materials needed for this process.
2. Sterilization: using moist heat, melt the agar pours in a boiling water at 121-134 degree C, to

kill all the variable spores and cleaning away all the unwanted microorganisms and then let it

cool do n to 85degree C. Molten agar solidifies temperature goes down below at 35 degree C.

3. Pouring and plating: sterilization should be immediately followed by plating that is slowly

pouring out to plate. This is to be carried out inside biosafety cabinet-sterile surface, atmosphere,

glassare and gands with alcohol.

4. Incubation: The period of the development of an infectious disease to take place.

Staining is done done for developing contrast and improving resolutions of the cells.

There are three basic techniques:

1. Simple Staining: one type of negative charged acidic or basic dye (positive charged) for

viewing the whole slide.

2. Differential Staining: this contains gram staining (the difference beween gram positive and

gram negative bacteria) and acid fast staining (distinguish amongst gram positive and gram

negative based on the presence or absence of waxy mycolic acid).

3. Structural Staining: this is based on chemical nature of specific organelle.