Gram staining

Perform Gram staining of the given bacterial smear

 

Objective: To perform Gram staining on a bacterial sample and differentiate between Gram-positive and Gram-negative bacteria based on color.

Theory: Gram staining is a widely used differential staining technique in microbiology that helps to classify bacteria into two major groups: Gram-positive and Gram-negative. In 1884, a Danish pathologist, Hans Christian Gram, discovered a method of staining bacteria using two dyes in sequence, each of a different color. he found that bacteria fall into two groups. The first group retains the color of the primary dye: crystal violet (these are called gram-positive). The second group loses the first dye when washed in a decolorizing solution but then takes on the color of the second dye, a counterstain, such as safranin  (these are called gram-negative). An iodine solution is used as a mordant (a chemical that fixes a dye in or on a substance by combining with the dye to form an insoluble compound) for the first stain.

The Gram stain is based on the ability of bacterial cell walls to retain the crystal violet dye during solvent treatment (decolorization). Gram-positive bacteria have a thick peptidoglycan layer that traps the crystal violet-iodine complex, resisting decolorization. In contrast, Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane, allowing the dye to wash out easily during decolorization and take up the counterstain

 Requirements:

• Bacterial sample
• Crystal violet (primary stain)
• Gram’s iodine (mordant)
• 95% alcohol or acetone-alcohol (decolorizer)
• Safranin (counterstain)
• Slides
• Distilled water
• Bunsen burner
• Inoculating loop
• Microscope
• Blotting paper

Procedure:

1.     Prepare a heat-fixed bacterial smear on a clean glass slide.

2.     Flood the smear with crystal violet and leave it for 1 minute.

3.     Rinse gently with distilled water.

4.     Flood the smear with Gram’s iodine and leave for 1 minute.

5.     Rinse gently with distilled water.

6.     Decolorize with 95% alcohol by adding drops until no more purple flows off (about 10-20 seconds).

7.     Immediately rinse with distilled water.

8.     Counterstain with safranin for 1 minute.

9.     Rinse with distilled water and blot dry with blotting paper.

10.  Examine under a microscope first using 10x objective, 40x and then 100x oil immersion lens.

 Observation:

S.N	Reagents used	Shape of bacteria	Color of bacteria	Inference

 

 

Result:

Gram-positive bacteria: Appear purple due to the retention of crystal violet stain. Gram-negative bacteria: Appear pink or red due to taking up the safranin counterstain.

 

Discussion:

The Gram staining procedure differentiates bacteria based on structural differences in their cell walls.

• Gram-positive bacteria have a thick peptidoglycan wall that traps the crystal violet-iodine complex, thus retaining the purple color even after alcohol decolorization.
• Gram-negative bacteria have a thin peptidoglycan layer and a high lipid content in their outer membrane; the alcohol dissolves the lipids and removes the primary stain, allowing the cells to take up the pink counterstain.
  This method is a preliminary test widely used in clinical laboratories to help identify bacterial pathogens and to guide initial antibiotic therapy.

Conclusion:

In this experiment, Gram staining was successfully performed on a bacterial sample. The staining technique allowed the differentiation of bacteria into Gram-positive and Gram-negative groups based on their cell wall properties. Gram-positive bacteria appeared purple, indicating their ability to retain the crystal violet stain, while Gram-negative bacteria appeared pink or red due to taking up the safranin counterstain.

Precautions:

• Prepare thin and even smears to avoid false results.
• Do not overheat during heat-fixing, as it can distort bacterial shapes.
• Proper timing for each staining and decolorizing step is crucial.
• Use fresh reagents for reliable results.
• Wash gently with water to prevent washing away the smear.
• Handle the microscope and glass slides carefully to avoid damage or accidents.

References:

1. Cappuccino, J. G., & Sherman, N. (2014). Microbiology: A Laboratory Manual (10th ed.). Pearson.
2. Pelczar, M.J., Chan, E.C.S., & Krieg, N.R. (1993). Microbiology: Concepts and Applications. McGraw-Hill.

  

Sterilization methods

 

APPLICATION OF DIFFERENT STERILISATION METHODS

Objectives:- To know about different sterilization methods used in the laboratory for tools, glassware, or other equipment to eliminate or reduce microbial contamination.

 Theory:

Sterilization is the process of eliminating or killing all forms of microbial life, including bacteria, viruses, fungi, and spores. It describes a process that destroys or eliminates all forms of microbial life. Sterilization is an absolute term, i.e., the article must be sterile, meaning the absence of all microorganisms. No relative terms like half sterile or semi-sterile can be used.

Various sterilization methods are used in hospitals, clinics, and laboratories to ensure the complete elimination of microorganisms from equipment, surfaces, or materials. There are some common sterilization methods like

1.     Red Heat 

Application: Red heat is commonly used in microbiology laboratories for sterilizing inoculating loops, needles, and other small metal instruments.

Process: The metal instrument is heated in a flame until it glows red, effectively killing any microorganisms present.

Use Cases: Used for routine sterilization of tools before and after use in microbiological procedures like streaking plates or inoculating cultures.

2.     Flaming:

Application:
Flaming is widely used in microbiology laboratories to sterilize the mouths of test tubes, culture tubes, flasks, and bottles, particularly during the transfer of microorganisms to prevent contamination.

Process:
The opening of the glass container (e.g., test tube or flask) is passed quickly through the flame of a Bunsen burner. The brief exposure to high temperature kills airborne or surface contaminants present around the mouth of the container.

Use Cases:

• Sterilizing the necks of culture tubes before and after taking or introducing inoculum.
• Commonly performed during aseptic techniques to maintain sterile conditions.
• Helps create an upward air current to prevent airborne microbes from entering the container.
• 
  
  

3.     Hot Air Oven:

Application: Hot air ovens are used for sterilizing glassware, metal instruments, and other heat-resistant materials that are not affected by high temperatures.

Process: Items are placed inside the oven and exposed to dry heat at temperatures typically ranging from 160°C to 180°C for a set period, usually 1 to 2 hours.

Use Cases: Commonly used in laboratories, pharmaceutical industries, and healthcare facilities for sterilizing equipment and glassware, as well as for decontaminating certain types of medical waste.

4.     Autoclave:

Application: Autoclaves are widely used in healthcare facilities, laboratories, and industrial settings for sterilizing a variety of materials, including glassware, surgical instruments, clothing, media, and equipment.

Process: The autoclave uses steam under pressure to achieve sterilization. Items are placed inside the autoclave chamber, and steam is introduced at high pressure 15 lb/in² and temperature (usually 121°C) for a specified duration, typically 15 minutes.

Use Cases: Essential for sterilizing heat-resistant materials that can withstand high temperatures and moisture. It is a crucial tool in medical and laboratory settings where absolute sterility is required.

Result and Discussion

Sterilization Method	Time	Temperature	Uses
Red Heat	A few seconds (until red hot)	~700–1000°C (flame)	Sterilizing inoculating loops, needles, and forceps tips
Flaming	2–3 seconds (brief exposure)	Flame temperature	Sterilizing the mouths of test tubes, flasks, and bottle openings
Hot Air Oven	1–2 hours	160–180°C	Sterilizing glassware, powders, oils, and metal instruments
Autoclave	15–20 minutes (standard)	121°C at 15 psi	Sterilizing culture media, surgical instruments, and laboratory waste

 

Bottom of Form

 

Dry heat sterilization is an effective method for achieving sterility in materials that can withstand high temperatures and do not require exposure to moisture. It is commonly used for sterilizing glassware, metal instruments, and heat-stable plastics in laboratory, healthcare, and industrial settings. While dry heat sterilization requires longer exposure times and higher temperatures compared to moist heat methods such as autoclaving, it offers advantages such as compatibility with heat-sensitive materials and ease of operation.

Moist heat sterilization, particularly through autoclaving, is a highly effective and widely used method for achieving sterility in various materials. By subjecting materials to high temperatures and steam under pressure, autoclaving rapidly and efficiently eliminates a wide range of microorganisms, including heat-resistant spores. This method is indispensable in laboratory, medical, and industrial settings for sterilizing equipment, media, glassware, surgical instruments, and other materials.

 

Conclusion: Hence, dry heat and moist heat methods were studied, that is employed in laboratory settings for the elimination of microorganisms.

 

Precautions:

Red heat

1. Allow the loop or needle to become red hot to ensure complete sterilization.
2. Cool the instrument before touching the culture to prevent killing the inoculum.
3. Hold the instrument at an angle to avoid splatter or aerosol formation.
4. Avoid heating near flammable materials or alcohol-based disinfectants.

Flaming

1. Pass the mouth of tubes or flasks briefly through the flame (2–3 seconds).
2. Do not overheat glassware—it may crack or explode.
3. Hold the tube at a 45-degree angle to reduce the risk of airborne contamination.
4. Ensure hands and clothing are away from the flame.

Hot Air Oven

1. Ensure the temperature and time are set properly (e.g., 160°C for 2 hrs).
2. Do not overload the oven; allow air circulation for even heating.
3. Use heat-resistant glassware and metal only.
4. Do not open the door immediately after the cycle ends—let it cool gradually to avoid breakage.

Autoclave

1. Ensure water level in the chamber is sufficient before use.
2. Do not seal containers tightly—steam must enter to sterilize properly.
3. Use autoclave tape or indicators to confirm sterilization.
4. Do not open until pressure is released completely and internal temperature drops.
5. Wear heat-resistant gloves and safety goggles while unloading.

Reference:

• Pelczar, M.J., Chan, E.C.S., & Krieg, N.R. Microbiology: Concepts and Applications, McGraw-Hill.
• WHO Laboratory Biosafety Manual, 3rd Edition.
• Cappuccino, J.G. & Sherman, N. Microbiology: A Laboratory Manual. Pearson Education.

 

Antibody, Immunoglobulin

 

                                               ANTIBODY                 By Anup Bajracharya

An antibody is a specialized protein produced by the immune system, specifically by B cells, in response to the presence of foreign substances called antigens. Antibodies are glycoprotein molecules called immunoglobulins. They react specifically with the antigen that induced their production.

Antibodies role:

• Recognizing and binding to foreign substances and facilitating their removal.
• Increasing phagocytosis.
• Neutralizing toxins or viruses.
• Activating complement.

Basic Structure

An antibody molecule has a basic structure (roughly Y-shaped) composed of four polypeptide chains connected by disulfide bonds. These four chains include two identical heavy (H) chains (the longer chains) and two identical light (L) chains (the shorter ones).Each chain contains a variable region (N-terminus) and a constant region (C-terminus).The four chains are assembled to form a Y-shaped molecule. Each light chain is attached to one heavy chain, and the two are attached to each other, all by disulfide bonds, to form a monomeric unit.

 

 

• Light chains:
• An immunoglobulin monomer contains two identical κ (kappa) or two identical λ (lambda) light chains, but never one of each.
• Each light chain usually consists of about 220 amino acids and has a molecular weight of 25000 daltons.
• They contain a variable (VL) domain and a constant (CL) domain.
• Each domain contains about 110 amino acids and an intrachain disulfide bond.
• The amino acid sequence in the VL domain is different between immunoglobulins synthesized by different B cells. The CL domain's sequence is constant.
• Heavy chains:
• There are five types of heavy chains: mu (µ), delta (ð), gamma (γ), epsilon (ϵ), and alpha (α).
• Each heavy chain consists of about 440 amino acids and has a molecular weight of about 50-70 kilodalton.
• Heavy chains contain one variable (VH) domain and three or four constant (CH) domains.
• The ð, γ, and α heavy chains contain three constant domains (CH1, CH2, CH3).
• The µ and ϵ heavy chains contain a fourth constant domain (CH4), making them both longer and heavier than γ, ð, or α heavy chains.

• When the antibody is cleaved at the hinge region with the papain enzyme, it gives two Fab fragments and one Fc fragment.

 

 Antigen-Binding Sites (Fab)

• The amino portion (NH terminus) of a single heavy and a single light chain form an epitope-binding site.
• A light chain variable domain (VL) and a heavy chain variable domain (VH) together form a cavity that constitutes the antigen (epitope)-binding region.
• Because an immunoglobulin monomer contains two identical light chains and two identical heavy chains, the two binding sites found in each monomeric immunoglobulin are also identical.

Fc Region

• The constant regions of the heavy chains for all molecules of the same immunoglobulin class have virtually the same amino acid sequence.
• This constant region (Fc region) is attached to the cell surface like Macrophages, PMNs, Monocytes, etc
• When the antibody is cleaved with papain enzyme, it gives one Fc fragment.

Immunoglobulin Isotypes/Classes

• IgG:
• Makes up the greatest amount of immunoglobulin in the serum (80% - 85%).
• Is the major immunoglobulin in extravascular spaces.
• Structure: Monomer with two γ heavy chains and two κ or λ light chains. Fc region has CH1, CH2, and CH3 domains.
• Is a 7S immunoglobulin with a molecular weight of 160 KDa.
• There are four human IgG subclasses: IgG1 (65-70%), IgG2 (23-28%), IgG3 (4-7 %), and IgG4 (3-4%).
• Many IgG antibodies effectively activate complement, opsonizing and neutralize microorganisms and viruses, and initiate antibody-dependent cell-mediated cytotoxicity (ADCC).
• Is the only class of antibodies that can cross the placenta from mother to fetus, mediated by a receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally well; IgG2 does not cross well.
• Fixes complement, though not all subclasses fix equally well; IgG4 does not fix complement.
• Macrophages, monocytes, PMNs, and some lymphocytes have Fc receptors for the Fc region of IgG.
• Produced later than IgM but provides long-lasting immunity.

 

 

• IgM:
• Found either as a cell surface-bound monomer (on unstimulated B cells) or as a secreted pentamer (5%-10 % of serum Ig).
• Is generally the first immunoglobulin to be formed following antigenic stimulation.
• Structure: Pentamer (secreted) or monomer (B cell surface).
• Pentamer consists of five monomers linked by disulfide bonds between heavy chains and a J Chain. All heavy chains in the pentamer are identical, as are all light chains. Each monomer has two μ heavy chains and two κ or λ light chains.
• 
  Secreted IgM is a 19S immunoglobulin. Monomeric IgM is 180KDa. IgM pentamer is more than five times larger than IgG.

• Each heavy chain has an additional domain (CH4) at the C terminus of the Fc region.
• IgM does not have a hinge region.
• The valence is theoretically 10 (in the pentameric form).
• Predominantly present intravascularly
• Is the first immunoglobulin made by the fetus or a virgin B cell upon stimulation.
• Is a good complement fixing immunoglobulin due to its pentameric structure.
• Is a good agglutinating immunoglobulin (immobilizing antigen), very efficient in clumping microorganisms.
• The multiple antigen-binding sites make it very efficient in functions like phagocytosis.
• Binds to some cells via Fc receptors.

 

• IgA:

• Present in both monomeric (in serum, 10% - 13% of serum Ig) and dimeric forms (in secretions).
• Serum IgA is a monomer (7S, 160 KDa).
• Secretory IgA (dimer) is found in mucosal surfaces and secretions.
• Dimer is formed by two IgA monomers joined by a J chain and also with a secretory component (SC).
• Epithelial cells transport the IgA dimer to mucosal surfaces using a specialized receptor, which becomes the SC.
• Secretory IgA dimers (11S, 360KDa) are found in mucus, saliva, tears, breast milk, and gastrointestinal secretions.
• IgA molecule has two α heavy chains containing three constant domains (CH1, CH2, CH3) and either two κ or two λ light chains.
• Important in local (mucosal) immunity.
• Normally does not fix complement, unless aggregated.
• Can bind to some cells (PMN's and some lymphocytes).
• Helps in phagocytosis and intracellular killing of microorganisms.
• Minor component in systemic humoral immunity, major role in mucosal immunity.
• IgA antibodies found in gut contents or feces are known as copro antibodies.

 

 

• IgD:
• (1% of serum Ig) has a monomeric structure. Found in low levels in serum; its role in serum is uncertain.
• Almost exclusively displayed on B-cell surfaces, where it functions as a receptor for antigen.
• IgD on B cells has extra amino acids at the C-terminal end for anchoring to the membrane.
• Does not bind complement.

 

 

• IgE:
• Present in relatively low serum concentration.
• Most is adsorbed on the surfaces of mast cells and eosinophils.
• Mast cells and basophils have specific receptors for the Fc portion of free IgE molecules.
• Mediates immediate hypersensitivity reactions (Type-1), such as anaphylactic shock, hay fever, asthma.
• Cross-linking of IgE on mast cell surfaces by antigen triggers the release of histamine and other inflammatory mediators.
• Involved in allergic reactions.
• Plays a role in parasitic helminth diseases. Measuring IgE levels helps diagnose parasitic infections.
• Does not fix complement.