Bacterial Cell Wall Staining by Chance's Method

  Bacterial cell is consisting of various structural components. Cell wall is one of the most important component. Cell wall present outside of the bacterial cell membrane. It gives rigidity, protection and shape to the bacterial cell.

  Based on the structure of cell wall, all bacteria are divided into two groups as -

  • Gram positive and
  • Gram negative.
Cell wall of Gram positive cell is monolayered while cell wall of gram negative bacteria is bilayered.
Bacterial cell wall can be demonstrated by various special staining methods like Chance's method, Ringer's method & Dyers method. The most common cell wall staining method is Chance's method.

Requirements :

  • Clean grease free slide
  • Nichrome wireloop
  • 24 hrs old culture of bacteria
  • 0.5% New fuchsin/Basic fuchsin solution (Basic Stain)
  • 0.5 % Condo red solution (Acidic Stain)

Procedure & Steps

  • Prepare the smear on the slide under aseptic conditions with the help of wire loop.
  • Air dry the smear, but do not heat fix (Because the heat fixation changes the structure of capsule. therefore heat fixation is avoided here).
  • Apply 0.5% New Fuchsin for 3 minutes.
  • Remove the excess stain (but do not water wash)
  • Apply 0.5 % Congo Red for 4 minutes.
  • Gentle wash with water
  • Air dry and observe under oil immersion objective lens.

Machanism and Principle of chance's cell wall staining method

  New Fuchsin stain is a basic stain. Therefore it is stained cell wall as well as cytoplasm. Cell wall and cytoplasm both are acidic in nature and they are having negative charge on their surface. However here the strong staining of cell wall take place. Because cell wall is more acidic than cytoplasm due to the presence of free carboxylic groups on its surface.

  Congo red is a acidic stain and this acidic stain bind with basic stain which is already present on the cell and removes that basic stain. That means here Congo red acts as a decolorizer. but the  removal of basic stain(New Fuchsin) takes place only from the cytoplasm and not from the cell wall, because it is strongly bound to the cell wall.

  Therefore after water wash cytoplasm becomes colourless while cell wall becomes pink coloured. that means here the role of Congo red is to carry out the decolorization, But the decolorization of only cytoplasm takes place and decolorization of cell wall does not take place.

Microscopic Observation

Spherical and rod shaped bacterial call wall Pink in colour.
 Under the microscope you will observed pink colour cell wall and colourless cytoplasm.

Gram Staining : Principle & Machanism, Steps, Disadvantages and Factors affecting gram Staining

Gram staining is a technique used to differentiate two major groups of bacteria based on their cell wall composition called gram positive and gram negative bacteria.

The technique was named after Hans Christian Gram who developed this method in 1884.
Gram staining is a preliminary test in the bacterial identification process. It plays an important role in clinical microbiology. It helps the medical professionals in the diagnosis of infectious diseases directly from the clinical sample.

The cell wall of gram positive bacteria is completely different from the gram negative bacteria. It is important for them to understand the Gram nature to provide the appropriate treatment for the infection.

Requirements of Gram Staining

  • Clean grease free glass slide
  • Nichrome wire loop
  • Bacterial cell suspension
  • 0.5% Crystal violet
  • Gram's lodine
  • 95% Ethanol
  • 0.5% Safranine/basic fuchsin

Steps of Gram Staining

Imagine our sample has a mixed culture with gram positive cocci and gram negative rods

Step-1 (Smear Preparation)

  • Prepare a thin smear of culture on a clean glass slide (oil free)
  • Allow to air-dry.

Step-2 (Heat Fixation)

  • Heat fix by exposing the smear to indirect flame
  • Heat fix slides above burner by moving slides through the peak of the flame.
  • Never heat fix wet slides !
  • Purpose of heat fixing is to kill and immobilize bacteria on slide.

Step-3 (Primary stain)

  • Add a few drops of Crystal violet on the smear.
  • Wait for a minute.
  • Wash the Crystal violet with water.
  • At this stage, all cells appear in purple colour.

Step-4 (Mordant)

  • Add plenty of Gram's lodine to the smear.
  • Wait for 1 minute.
  • Rinse with water.
  • Cells appear to be purple yet.

Step-5 (Decolourising)

  • Decolourise with 95% Ethyl Alcohol for not more than 30 seconds.
  • Wash with water.
  • At this stage, gram positive cells appear in purple colour And gram negative appear as colourless.

Step-6 (Counter stain)

  • Add counter stain Safranin to the smear.
  • Wait for 1 minute.
  • Wash with water.

Step-7 (Microscopic Observation)

  • Dry the slide and put a drop of immersion oil on the smear & Observe under 100X lens.

  • Gram positive cells retain the purple colour
  • Gram negative cells appear in pink colour.

Machanism of Gram Staining (Principle) :

The widely accepted theory is based on the differences in the cell wall composition between the gram-positive and gram-negative bacteria.

When crystal violet and iodine are added to the smear both will penetrate through the cell wall. And form a large crystal violet iodine complex (CVI complex) within the inner and outer layers of gram positive and gram negative bacteria.

The cell wall of gram-negative bacteria is thin and made of one or two layers of peptidoglycan. In addition to this it has got an outer lipopolysaccharide layer surrounding the cell wall.

  When a decolorizer like alcohol or acetone is added. The outer lipopolysaccharide layer will be completely dissolved leaving the thin peptidoglycan layer exposed.
The effective alcohol makes the peptidoglycan layer become perforated.

  In the decolorization step the gram-negative bacteria failed to retain the CVI complex and become colourless as the complex is washed away.

On contrary to this thick and multi-layered peptidoglycan in gram positive bacteria will be dehydrated by the addition of alcohol. Because of the thick peptidoglycan layer and dehydration by the alcohol treatment the CVI complex gets trapped in the cell.

  Therefore after the decolorization step the gram positive bacteria appear purple in color and the gram negative bacteria become colorless in the last step.

  When a counter stain likes a safranine is added the gram negative bacteria easily absorbed the stain and gives pink color to the cells. The gram positive bacteria remain purple in color due to the retention of CVI complex which is darker than Safranine.

Factors affecting Gram staining

  • Age of culture.
  • Excessive heat fixation.
  • Thick smear or overcrowding of cells.
  • Old staining reagents.
  • Air drying.
  • Over decolourization.

Disadvantages of Gram Staining

Athough the gram staining is used as primary test in the identification process this method will not be able to identify the bacteria to the species level. It will be used in combination of other modern and traditional identification tests
  The CVI complex may get lost from the gram positive bacteria due to over decolorization, which might lead to misinterpretation.
  There were evidences where some old gram positive cultures were not able to retain purple color and therefore observe as gram negative. 

Steps Involved in Transcription

 Transcription is a heterocatalytic function of DNA which involves transfer of coded information from DNA through the synthesis of RNA over the template of DNA. Only one of the strand of DNA called sense strand (master strand) is involved.
  The segment of DNA involved in transcription is cistron. It has a promoter region where initiation begins and a terminator region where transcription ends.

Enzyme involved in transcription is RNA polymerase. It is single in prokaryotes. There are three types of RNA polymerase in eukaryotes,
I for 28s, 18s & 5.8s RNA.
II for mRNA and SnRNA and
III for tRNA, 5s RNA & Sc RNA.

RNA polymerase has five polypeptides — σ, α, β, β' and ω).

σ or sigma factor recognises the promoter region while the remaining or core enzyme takes part in transcription.
A rho or ρ factor is needed for termination of transcription.

Steps involved in transcription

a] Activation of Ribonucleotides :

With the help of phosphorylase, energy and phosphoric acid, ribo- nucleoside monophosphates are changed into triphosphates ATP, GTP, CTP & UTP.

b] Cistron :

It has a promoter region where transcription begins and a terminator region where transcription ends.
Promoter region possesses RNA polymerase recognition as well as RNA-polymerase binding sites.
Sigma factor or RNA polymerase gets attached to promoter region of cistron. The two DNA strands separate with the help of unwindase and helix destabilising proteins.
One of them function as sense strand and takes part is transcription. Transcription proceeds in 5' to 3' direction.

c] Base Pairing :

Ribonucleoside triphosphate come to lie opposite complementary nitrogen bases of sense strand (A opposite T, C Copposite G, U opposite A and G opposite C).
Pyrophosphatase hydrolyses two phosphates from cach activated nucleotide. This releases energy.

d] Strand Formation : 

RNA polymerase establishes phosphodiester bonds between adjacent ribonucleotides.
Sigma factor σ recognises.
Core enzymes moves along the sense strand till it reaches terminator region and separates from DNA template in the presense of rho (ρ) factor.
Terminator region contains a stop signal made of 4-8 A (poly A tail) nucleotides.

e] Separation of RNA :

rho (ρ) factor has ATP ase activity. It helps in separating the newly formed RNA or primary transcript from the sense strand of DNA.

f] Duplex Formation :

With the release of primary transcript the sense and antisense strands of DNA re-establish their hydrogen linkages and form duplex. Unwindase and helix destablising proteins are released.

g] Post-Transcriptional Processing :

Primary transcript is often called heterogenous or hn RNA as it is generally bigger than the functional RNA.
  1. Cleavage : hn RNA is broken to form smaller pieces. Ribonuclease-P (RNA enzyme) separates 5-7 t RNA precursors from primary transcript.
  2. Splicing : Introns or intervening sequences of nonessential nature are removed by nucleases. Ribozyme (RNA enzyme) is one such enzyme.
  3. Terminal Additions : Nucleotides are added at the ends of RNA for specific functions, e.g. CCA at 3'ends of tRNA, cap nucleotides at 5'ends of m RNA or poly A nucleotides at 3'end of m RNA.
  4. Nucleotide Modifications : Certain nucleotides are methylated, ethylated, deaminated, etc. to produce different chemicals like inosine, methyl cytosine, dihydrouracil, pseudouracil, etc.

The promoters of prokaryotic genes are simple. They generally consist of about 40 bp of DNA that contain two elements :

  • a TATA box, located 10 bp upstream from the transcription initiation site and
  • a second element, located 35 bp in the 5' direction upstream.

Eukaryotic promoters are more complex and usually consist of several elements. One set of elements ensures that transcription initiates at the correct site. The elements in this set, which of ten include a TATA box, which are usually found within 100-200 bp of transcription start site. Another set of clements, usually located further 5' from the transcription initiation site, can enhance or silence transcription. These work with, or serve as tissue specific or signal response elements.

Ribosomal RNA is also synthesized as a large 45s precursor molecule that is processed into the mature 28s, 18s and 5.8s fomms.

Metabolism: Definition, Terminology, Feaction & Function

The metabolism applies to the assembly of biochemical reactions which are employed by the organisms for the synthesis of cell materials and for the utilization of energy from their environments.

  Metabolism may be defined as 'the sum total of all the enzyme-catalyzed reactions that occur in an organism'.

  The large number of reactions in a cell are organized into a relatively small number of sequences or pathways. It is a highly coordinated and purposeful cell activity, in which multienzyme systems co-operate. This obviously points out to the fact that the metabolism of even a simple unicellular organism is time variant.

Metabolism in microorganisms and the human beings:

a] Microorganisms like bacteria (e.g., Escherichia coli) can double in number every 40 minutes in a culture medium containing only glucose and inorganic salts, or in 20 minutes in a rich broth. 

  The components of the medium are depleted and very little is added to the medium by the cells. Each cell contains hundreds to thousands of molecules of each of about 2,500 different proteins, about 1,000 types of organic compounds and a variety of nucleic acids. It is, thus, apparent that the bacterial cells participate in a variety of metabolic activities.

b] Human adults maintain a constant weight for about 40 years, during which period a total-~1! about 60 quintals of solid food and 45,000 litres of water are metabolised. And yet boll body weight and body composition remain almost constant.

TERMINOLOGY OF METABOLISM

The various processes constituting metabolism may be divided, somewhat arbitrarily, into catabolism and anabolism. Those processes, whose major function is the generation of chemical energy in forms suitable for the mechanical and chemical processes of the cells, are termed as catabolism; whereas those processes, which utilize the energy generated by catabolism for the biosynthesis of cell components, are termed as anabolism.

The various activities powered by catabolism include mechanical movement, growth, reproduction, accumulation of foods, elimination of waste, generation of electricity, maintenance of temperature etc. The various anabolic activities may be exemplified by food manufacture etc.

Some processes can be either catabolic or anabolic, depending on the energy conditions in the cell. These are referred to as amphibolism.

Catabolism reaction
Fuels(carbohydrates, fats) ➞ CO₂+ H₂O + Useful energy
Anabolic reaction
Useful reaction + Small molecules ➞ Complex molecules

FUNCTION OF METABOLISM

In all cells, metabolism enables the cell to perform its vital functions. Metabolism performs following 4 specific functions:
  1. to obtain chemical energy from the degradation of energy-rich nutrients or from captured solar energy.
  2. to convert nutrient molecules into precursors of cell-macromolecules.
  3. to assemble these precursors into proteins, lipids, polysaccharides, nucleic acids and other cell components.
  4. to form and degrade biomolecules required in specialized functions of cells.

Metabolism are closely interrelated since the synthesis of the molecules, that are a component of cell, requires an input of energy, while at the same time it is obvious that the cell components are needed to provide the energy supply and to control intracellular solute concentrations.

The specialized functions also require biosynthetic processes as well as a supply of energy. Terms catabolism and dissimilation are synonyins and refer to the pathways or routes breaking down food materials into simpler compounds and resulting in the release of energy contained in them.

The processes of anabolism or assimilation utilize food materials and energy to synthesize cell components.

The energy relations of the biological processes, the term excrgonic is used to denote a chemical reaction which liberates chemical-free energy. The term exothermic refers to the total energy liberated including heat.

  As the magnitude of heat energy is small and also that it cannot drive biological reactions, the biochemists are more interested in free energy changes and often use drive term exergonic.

The corresponding energy-consuming term endergonic refers to the processes which require an input of free energy while the term endothermic denotes a total energy requirement (including heat).

The Five Kingdom Classification by Robert Whittaker & It's Limitations

 Robert Whittaker (1969), proposed the first popular classification system called five-kingdom system, which was accepted widely. The classification system is based on three criteria.

1. Cell types

  1. Prokaryotie (cells with primitive nucleus and lacking membrane enclosed organelles) and
  2. Eukaryotic (cells with well-developed nucleus and membrane-enclosed organelles.

2. Level of cellular organization 

  1. Unicellular or
  2. Multicellular.

3. Mode of nutrition

  1. Photoautotrophic nutrition, which is concerned with use of sunlight as energy source and CO2 as source of carbon.
  2. Heterotrophic nutrition, which is concerned with use of organie compounds as source of energy and carbon. It may be absorptive (which means absorption of nutrients by body wall) and
  3. Ingestive (which means intake of solid food particles ).

  This system consists of one prokaryotic kingdom of Monera (which are prokaryotes) and four eukaryotic kingdoms - Protista, Fungi, Plantae and Animalia.

The Five Kingdom Classification  by Robert Whittaker

I] Kingdom Monera (Prokaryotes)

  • It includes bacteria, cyanobacteria and archaebacteria.
  • Unicellular, microscopic, solitary or colonial forms.
  • Can respire aerobically or anaerobically and reproduce by asexual, sexual or vegetative methods.
  • Act as decomposers and mineralisers and some may be concerned with nitrogen fixation also.
  • No membrane bound organelles like mitochondria and Golgi complex. No nuclear membrane.
  • Mode of nutrition: Nutritionally. these organisms exhibit great diversity from autotroph to heterotroph, phototroph to chemotroph and organotroph to lithotroph.

II] Kingdom Protista (Unicellular Eukaryotes)

  • Usually (phytoplanktons or zooplanktons).
  • Organisation ranges from unicellular to multicellular colonial forms. aquatic pue planktonic
  • Some are commensals and some are parasites (heterotrophic or absorptive mode of nutrition)
  • Some coloured algae contain various types of accessory pigments.
  • Reproduction is by asexual as well as sexual modes and both haploid as well as diploid forms exist.
  • Locomotion pseudopodial, ciliary or flagellar depending on the type of locomotary appendages present.
  • The phytoplanktons are producers whereas the zooplanktons are consumers (holozoic nutrition).

III] Kingdom Fungi (Multicellular Decomposers)

  • Include non-green plants that are the important decomposers and mineralizers.
  • Hyphae constitute an entangled cottony mass of filaments termed mycelium that may be aseptate coenocytic or septate multicellular.
  • Possess a cell wall made up of chitin or fungal cellulose and membrane bound organelles.
  • May be solitary unicellular types or multicellular filamentous types called hyphae.
  • Plastids are absent.
  • Mode of nutrition heterotrophic (absorption and extracellular digestion), saprophytic or parasitic.
  • Non-motile in forms but some may produce motile zoospores.
  • Embryo formation does not occur but various fruiting bodies are formed.
  • Most members pathogenic on plants as well as animals and cause various diseases as rust, smut and mildew (in higher plants).
  • Useful fungi include yeast, Penicillium etc. used in bakery, brewery and antibiotic industries.
  • Intercellular mycelia give out haustoria that absorb the nutrients from host cells.
  • Asexual and sexual reproduction observed: members with no sexual reproduction known fungi imperfecti.
  • Edible fungi: mushrooms and morels.

IV] Kingdom Plantae (Multicellular Producers)

  • Members consist of muiticellular, green, photosynthetic, primary producers of the biosphere.
  • Aquatic as well as terrestrial, large-bodied and non-motile (except bryophytes which produce motile zoospores).
  • Cells bound by cellulosic cell wall and contain the photosynthetic pigments or chloroplast.
  • Higher plants have complex cellular organization and vascular tissues (tracheophytes).
  • Reproduction: by vegetative and sexual methods.
  • All plants produce seeds except bryophytes and pteridophytes.
  • Nutrition: autotrophic, absorptive (insectivorous plants) or parasitic (totally parasitic e.g. Cuscuta or partial parasite, e.g. Santalum).
  • Lower plants possess simple thalloid organization and lack vascular tissue (bryophytes).

V] Kingdom Animalia (Multicellular Consumers)

  • Consist of multicellular, eukaryotic organisms, lacking cell wall, plastids and photosynthetic pigments.
  • Nutrition: by ingestion and subsequent digestion within the gastric cavity some possess absorption type of nutrition (parasites).
  • Reproduction: asexual or sexual in lower forms while predominantly sexual in higher forms.
  • Animals: main consumers in the food chain and may be primary consumers (herbivores) or secondary consumers (carnivores).
  • Bear locomotory appendages and are, therefore, motile.
  • Characterized by well developed muscular and nervous systems.

Limitations of Five Kingdom System

Most microbiologists do not accept the five-kingdom system for following reasons.
  1. It includes prokaryotes in Monera, but it does not distinguish between Bacteria and Archaea.
  2. There is a great diversity in kingdom Protista. Protozoa are heterotrophs with animal like cell organization. Algae are photoautotrophs with plant like cell organization.
  3. Boundaries between Protista, Plantae and Fungi are poorly defined. For example, the red algae is not closely related to plants, still it is included in Plantae.
  4. It does not explain about evolution and phylogenetic relationship.

How Does 70% Alcohol-based Hand Sanitizers Kill Microbes ?

Hand sanitizers often contain alcohol such as ethyl alcohol as an active ingredient other ingredients may include water and glycerin. Hand sanitizer has reduced the levels of microorganisms by killing them, just like other disinfectants do. 

  The effectiveness of hand sanitizers depend on quantity used duration of exposure and frequency of use. Usually 70% alcohol-based hand sanitizers, if rubbed thoroughly over finger and hand surfaces for 30 seconds followed by complete air drying can effectively reduce populations of bacteria, fungi and some viruses.

How alcohol-based hand sanitizers kill microorganisms ?

  • Alcohols disturb the normal arrangement of lipids structure of bacterial plasma membrane. The plasma membrane of the bacteria is formed by several phospholipid molecules.
  • Phospholipid is composed of a phosphate group and a lipid. The phosphate group is hydrophilic and the lipid is hydrophobic.
  • The phospholipid bilayer of plasma membrane which is formed by two layers of phospholipids is arranged in such a way that the hydrophilic parts are directly exposed to the internal and external environments of the cell. 

Plasma membrane of microorganisms

  • The reason being is that the internal and external environments of the cell are occupied with water.
  • In most cases the hydrophilic ends are always in contact with water. The hydrophilic nature provides the membrane more stability in the aqueous solutions.
  • When phospholipids come in contact with alcohol, the lipid molecules move out of place and break the arrangement as the hydrophilic ends chase for water. Thus it increases the membrane fluidity.
  • This alcohol stressed condition changes the shape of proteins that are present on the membrane.
  • When the proteins are not in normal shape they cannot perform their routine job. Therefore the microorganisms cannot survive.

Hand sanitizers are commonly used in pharmaceutical companies, hospitals and clean rooms.
Particularly alcohol-based hand sanitizers will be used in combination of water.

Why Alcohol solution with 70% alcohol and 30% of water effectively kills bacteria ?

  • Alcohol in combination of water evaporates slowly, therefore increases the contact time of the sanitizer and thus works effectively.
  • Water acts as a catalyst and plays an important role in denaturing the proteins.
  • The 100% alcohol coagulates the proteins rapidly and forms a protein layer. This will prevent coagulation of other protein molecules. This is the reason why the 70% alcohol is found to be more effective than 100%.

Prokaryotic Ribosome: structural subunits and function

 Ribosomes present in all living cells. Ribosomes are made up of several RNAs and proteins. They act as site of protein synthesis where mRNA translation takes place. Ribosomes link each amino acid in the order specified by the codons on mRNA to form a polypeptide chain.

Bacterial ribosome structure

  • Several ribosomes come together to form polysome during the protein synthesis. The rate of protein synthesis depends on the number of ribosomes.
  • Bacterial cell contains about     10000 ribosomes and they contribute 30% of total dry weight of the cell.
  • In Bacterial cell ribosomes are present in the cytoplasm.
  • More number of ribosomes provide granular appearance to the cytoplasm.
  • Ribosomes are of two types 70s and 80s.
  • The 80s ribosomes are present in eukaryotic cells.
  • Bacterial cell contains 70s ribosomes
  • Here the "s" denotes svedberg units.
  • Svedberg units indicate the rate of sedimentation during ultracentrifugation.
  • The sedimentation rate depends on size, shape and weight of the molecule, for example svedberg value is more for the heavier molecules than the lighter molecules.
  • These units are named after Theodor Svedberg from sweden who discovered the principle of ultracentrifugation. He was awarded the nobel prize in 1926.

Structure of 70s Ribosome

  • The 70s ribosome is made of two subunits a small and a large subunit.
  • Smaller subunit is of 30s and the larger one is a 50s.
  • Similarly the 80s ribosomes in eukaryotic cell consist of a 40s and 60s subunits.

30s Subunit of Ribosome

  • The smaller subunit in 70s ribosome(30s) has 16 sRNA and comprise of 1540 nucleotides bound to 21 proteins.

50s Subunit of Ribosome

  • The larger subunit has 5 sRNA and 23 sRNA.
  • The 5sRNA comprise of 120 nucleotides bound to 31 proteins.
  • The 23sRNA comprise of 2900 nucleotides bound to the 31 proteins.

Protein synthesis by Ribosome

  • During the protein synthesis the mRNA binds to the smaller subunit(30s).
  • The larger subunit(50s) links amino acids by providing the peptide bonds to the polypeptide chain.
  • The two subunits are combined together. The strength of the attachment depends on the concentration of Mg++ ions.