Pure culture techniques-Streak, Spread, pour plate methods

Isolation of Pure Cultures

In natural habitats microorganisms usually grow in complex, mixed populations with many species. This is a problem for microbiologists because a single type of microorganism cannot be studied adequately in a mixed culture. A pure culture which is a population of cells arising from a single cell is needed to characterize an individual species. 

Pure cultures are so important that the development of pure culture techniques by the German bacteriologist Robert Koch transformed microbiology. Within about 20 years after the development of pure culture techniques most pathogens responsible for the major human bacterial diseases had been isolated.

There are several ways to prepare pure cultures. Pure cultures usually are obtained by isolating individual cells with any of three plating techniques: the spread-plate, streak-plate and pour-plate methods.

The spread-plate and pour-plate methods usually involve diluting a culture or sample and then plating the dilutions. In the spread-plate technique, a specially shaped rod is used to spread the cells on the agar surface; in the pour-plate technique, the cells are first mixed with cooled agar-containing media before being poured into a petri dish.

The streak-plate technique uses an inoculating loop to spread cells across an agar surface.

 

The Spread Plate and Streak Plate

    If a mixture of cells is spread out on an agar surface at a relatively low density, every cell grows into a completely separate colony, a macroscopically visible growth or cluster of microorganisms on a solid medium. Because each colony arises from a single cell, each colony represents a pure culture.

 The spread plate is an easy, direct way of achieving this result. A small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod. The dispersed cells develop into isolated colonies. Because the number of colonies should equal the number of viable organisms in the sample, spread plates can be used to count the microbial population.

 

    Pure colonies also can be obtained from streak plates. The microbial mixture is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several patterns. After the first sector is streaked, the inoculating loop is sterilized and an inoculum for the second sector is obtained from the first sector. A similar process is followed for streaking the third sector, except that the inoculum is from the second sector. Thus this is essentially a dilution process. Eventually, very few cells will be on the loop, and single cells will drop from it as it is rubbed along the agar surface. These develop into separate colonies. In both spread-plate and streak-plate techniques, successful isolation depends on spatial separation of single cells.

 

 

The Pour Plate

Extensively used with procaryotes and fungi, a pour plate also can yield isolated colonies. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating. Then small volumes of several diluted samples are mixed with liquid agar that has been cooled to about 45°C, and the mixtures are poured immediately into sterile culture dishes. Most bacteria and fungi are not killed by a brief exposure to the warm agar. After the agar has hardened, each cell is fixed in place and forms an individual colony. 

Like the spread plate, the pour plate can be used to determine the number of cells in a population. Plates containing between 30 and 300 colonies are counted. The total number of colonies equals the number of viable microorganisms in the sample that are capable of growing in the medium used. Colonies growing on the surface also can be used to inoculate fresh medium and prepare pure cultures.

These techniques require the use of special culture dishes named petri dishes or plates after their inventor Julius Richard Petri, a member of Robert Koch’s laboratory; Petri developed these dishes around 1887. They consist of two round halves, the top half overlapping the bottom. Petri dishes are very easy to use, may be stacked on each other to save space, and are one of the most common items in microbiology laboratories.

Gene therapy

 Genes are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders develops. 

Gene therapy is a technique for correcting defective genes responsible for disease development. Several approaches can be used for correcting faulty genes:

·   A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.

·        An abnormal gene could be replaced with a normal gene through homologous recombination.

·        The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

·        The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA.

Target cells such as the patient's liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

Gene therapy may be classified into two types:

Germ line gene therapy

Here germ cells, (sperm or eggs) are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations.

Somatic gene therapy

Here the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited into later generations.

·       Some of the different types of viruses used as gene therapy vectors are Retroviruses (A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells), Adenoviruses (A class of viruses with double-stranded DNA genomes), Adeno-associated viruses (A class of single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome), Herpes simplex viruses (A class of double-stranded DNA viruses), etc.

·       Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.

·       Another nonviral approach involves the creation of an artificial lipid sphere with an aqueous core (liposome). This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane.

·       Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors and get into the interior of the target cell.

·       Introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations. A problem with this method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

Gene Therapy in India

Gene Therapy can be a viable option for increasing the regeneration of the disease for which no other reliable treatment is available. The number of injections required for treating most of the conditions is comparatively low and costs less than the conventional alternatives, The advantages include long-term effects such as the possibility of permanent solutions for the conditions treated by gene therapy. The removal of problematic genes from the body of future parents also removes any chances of recurrence of the same condition in the next generation also.  Gene Therapy can treat Parkinson’s disease, muscular dystrophy, Kidney problems, eye diseases, neurodegenerative Diseases and Immune deficiencies.

Gene therapy in India is rapidly advancing, marked by the launch of affordable, indigenous CAR-T cell therapy for cancer (NexCAR19) from IIT Bombay in 2024.. The ICMR and DBT provide regulatory guidance, to make these costly therapies accessible and affordable for India's large patient population. The focus is on oncology, rare diseases, and genetic conditions like Muscular Dystrophy.

·        Cancer (CAR-T - Chimeric antigen receptor T cell Therapy):  India launched its first indigenous CAR-T cell therapy, NexCAR19, for B-cell malignancies, making advanced cancer treatment affordable.

T cells are the backbone of CAR T-cell therapy. Collect blood from the patient and separate out the T cells. These cells are then genetically engineered to produce special proteins on their surfaces called chimeric antigen receptors, or CARs. The CARs help the cells to bind on to specific antigens, that are present on cancer cells (and some normal cells). They enhance the T cells' ability to kill cancer cells. These modified T cells are grown, and returned to the patient as a single infusion. Currently, this entire process—from the initial blood collection to the cells being infused back into the patient—takes about 3 to 5 weeks. T cells will grow in the patient's body and, bind to cancer cells using their special receptors killing them.

·        Hemophilia A: A landmark trial showed successful gene therapy, eliminating the need for regular infusions by enabling patients to produce Factor VIII, offering a long-term solution.

·        Rare Diseases Focus: India is actively developing gene therapies for numerous rare genetic disorders, addressing a significant unmet need for conditions like Muscular Dystrophy, night blindness, and sickle cell anemia.

Disadvantages:

• Short-lived nature of gene therapy - The therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits.
• Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system that reduces gene therapy effectiveness is always a potential risk.
• Problems with viral vectors – Viruses may cause toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
• Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be difficult to treat effectively using gene therapy.

PERMANENT SLIDE PREPARATION

 

A permanent slide is a prepared microscope slide that can be stored for a long time without deteriorating. It is commonly used in biological and medical laboratories for the observation of specimens, such as cells, tissues, microorganisms, and other biological samples. The preparation of permanent slides involves the following basic steps:

Steps in Permanent Slide Preparation:

1. Fixation: Preserves the specimen structure and prevents decomposition.
2. Dehydration: Removes water from the specimen to prepare it for the mounting medium (especially if using organic solvents like Canada balsam or for specimens that will be mounted with resins).
3. Clearing: Makes the specimen transparent and prepares it for embedding in the mounting medium.
4. Staining: Adds color to specific parts of the specimen (e.g., cells, tissue, bacteria) to improve contrast for microscopic observation.
5. Mounting: Involves placing the specimen in a mounting medium to preserve and protect it long-term.
6. Sealing: Seals the specimen and mounting medium under a cover slip.

 

Materials Needed:

• Glass slide: Standard microscope slide.
• Cover slip: Thin glass cover (round, square, or rectangular) that protects the specimen and provides a flat surface for observation.
• Specimen: The biological sample to be observed (e.g., tissue, microorganisms, or cells).
• Fixative: Chemical solutions used to preserve the specimen, such as formaldehyde, ethanol, or acetic acid.
• Staining agents: Dyes used to color the specimen, such as Gram stain, Methylene blue, Iodine, Safranin, or Hematoxylin.
• Mounting medium: A liquid medium that helps to preserve the specimen and provide clarity for viewing (e.g., glycerin, Canada balsam, or DPX).
• Forceps: For handling small specimens and cover slips.
• Dissecting needle or brush: To handle small specimens, especially for bacterial slides.
• Distilled water: For rinsing specimens during preparation.

 

Steps for Preparing a Permanent Slide:

1. Collection and Preparation of Specimen

• Collection: Obtain a small sample of the specimen to be examined.
• Cutting (for tissue samples): If preparing a tissue sample, cut it into thin sections using a sharp scalpel or razor blade. The thickness of the sections depends on the specimen and the desired observation level (usually around 5–10 microns for tissues).
• Air-drying (for wet specimens): If the specimen is moist, allow it to air-dry for a few minutes.

 

2. Fixation

·        The fixation process preserves the specimen and maintains its structure by killing cells and microorganisms and preventing decomposition. A good fixative penetrates tissue quickly, reduces the shrinkage, and does not change the biological components of the tissue. The fixation of two types:

·        Physical fixation: by heating or cooling to the extent of freezing.

·        Chemical fixation: by using chemical fixative like formaldehyde in light microscope. In the electron microscope different fixative is used such as glutaraldehyde and potassium permanganate ( KMNO4).

Depending on the specimen, choose an appropriate fixative. For example:

• Formalin (10% formaldehyde) is commonly used for tissue specimens.
• Alcohol (e.g., ethanol) is used for microorganisms.
• Acetic acid is used for some types of plant material.
• Procedure:
• Place the specimen on the slide and add a few drops of the fixative.
• If using a tissue sample, immerse it in the fixative for 10–20 minutes. For microorganisms, a quick immersion (1–2 minutes) is usually sufficient.
• Rinse: After fixation, rinse the specimen with distilled water to remove excess fixative.

·        The goal of this process is to maintain the composition of cells and preventing the growth of bacteria in the sample and prevent decomposition also leads to hardening samples and make it possible to cut into thin slices.

3. Dehydration and Clearing

·        For long-term preservation, the slide must be properly dehydrated. The sample is exposed to an escalating series of alcohols like ethanol starting from 50%, 70%, 90%, 100% alcohol to gradually remove water from the specimen.  After dehydration, clearing agents like xylene or toluene are used to make the specimen transparent, allowing the mounting medium to flow easily and preserve clarity. Add a drop of xylene or toluene to the specimen and let it sit for a few minutes. Clearing is the process of making the sample or section clear and free of impurities and remove the dehydration solutions.

 

4. Staining

Staining enhances the contrast of the specimen, making it easier to observe under the microscope. Staining in the light microscope are by different dyes such as Eosin, Hematoxylin, Safranin, Toluidine blue while in Electron Microscope by uranyl acetate or lead citrate.

• Prepare Staining Solution: Depending on the specimen, use an appropriate dye or stain:
• For bacteria: Gram stain or Methylene blue.
• For plant cells: Iodine or Safranin.
• For animal tissues: Hematoxylin and Eosin (H&E).
• Staining Process:
• Place the slide with the specimen on a staining tray or dish.
• Add a few drops of the staining solution and let it sit for a period (usually 1-2 minutes for bacterial specimens, and up to 10 minutes for tissue specimens, depending on the stain).
• After staining, rinse the slide gently with distilled water to remove excess stain.
• Dehydration: If the specimen needs to be dehydrated (as with some histological preparations), pass it through a series of alcohol solutions (50%, 70%, 95%, 100%) to remove water from the sample. This step may be necessary for certain tissues, especially when using thicker stains like hematoxylin.

 

8- Mounting

Mounting involves placing the specimen in a mounting medium that will help preserve it and provide a medium that will support the cover slip. A drop of resinous substance such as Canada balsam, DPX, or PVA, is placed on the slide and covered with a glass cover and the slide is saved until the study.

 

• Common mounting media include:
• Glycerin: Used for temporary mounts or when long-term preservation is not necessary.
• Canada balsam: A commonly used resinous medium for permanent mounts. It’s clear, hardens over time, and provides excellent transparency.
• DPX (Distyrene Plasticizer Xylene) is another permanent mounting medium often used in histology.
• Place the Cover Slip: Gently lower a cover slip over the specimen using forceps. Be careful not to trap air bubbles between the slide and cover slip.
• Press Gently: Apply gentle pressure to the cover slip to spread the mounting medium evenly and expel excess fluid.

9. Sealing the Slide

To ensure the specimen remains securely mounted for long-term observation, the edges of the cover slip are sealed with a sealant.

• Sealant Application: After the cover slip is in place, apply a small bead of clear nail polish, paraffin wax, or Permount around the edges of the cover slip. This prevents the mounting medium from evaporating and helps to keep the cover slip in place.
• Allow the slide to dry for several hours to ensure that the sealant hardens.

11. Storage and Labeling

Once the slide is prepared and sealed, it can be stored for future use:

• Labeling: Write the name of the specimen, the preparation date, and any relevant details on the slide or slide holder (using a permanent marker or label).
• Storage: Store permanent slides in a cool, dry place. Ideally, slides should be kept in a slide box or slide rack to prevent contamination and damage.

For histological sections, embedding and sectioning can also be done.

Embedding

Paraffin wax is used to embed the samples to be examined by light microscope. After immersion in the wax, sample is poured in templates and left to cool and solidify.  In the electron microscope embedding material used must be more solid and more resistant to temperatures.

Sectioning

Sectioning is the process of getting thin slices of a certain thickness, using mechanical or manual devices called microtome. Microtome is a tool used to cut extremely thin slices of material, known as sections. Important in science, microtomes are used in microscopy, allowing for the preparation of sample for observation under transmitted light or electron radiation. Microtomes use steel, glass, or diamond blades depending upon the specimen being sliced and the desired thickness of the sections being cut. Steel blades are used to prepare sections of animal or plant tissues for light microscope histology, Glass knives are used to slice sections for light microscopy and to slice very thin sections for electron microscope. Industrial grade diamond knives are used to slice hard materials such as bone, teeth and plant matter for both light microscopy and for electron microscopy. Diamond knives are used for slicing thin sections for electron microscope.

 There are many types of microtome such as Sledge microtome for the preparation of large samples, such as those embedded in paraffin for biological preparations, Rotary microtome for hard materials, such as a sample embedded in a synthetic resin, Cryo-microtome for the cutting of frozen samples, Ultramicrotome can allow for the preparation of extremely thin sections, Vibrating microtome is usually used for difficult biological samples. Saw microtome is especially for hard materials such as teeth or bones. Laser microtome can also be used for very hard materials, such as bones or teeth as well as some ceramics.

Conclusion

Permanent slide preparation is a process that requires attention to detail to ensure the specimen is properly preserved, stained, and mounted for long-term observation. By following the proper steps of fixation, staining, dehydration, clearing, and mounting, the slide can be kept intact for years, making it useful for further study or reference. This process is widely used in microbiology, histology, and other biological fields.

 

 

Fungal Staining

Fungi are eukaryotic organisms with both macroscopic and microscopic characteristics.  Lactophenol Cotton Blue (LPCB) Staining is a simple histological staining method used for the microscopic examination and identification of fungi. The fungal spore cell wall is made up of chitin, which, the components of the Lactophenol Cotton Blue solution stains for identification. The lactophenol cotton blue solution acts as a mounting solution as well as a staining agent. The solution is blue in color and it is made up of a combination of three main reagents:

·        Phenol: It acts as a disinfectant by killing living organisms

·        Lactic acid: It preserves the fungal structures

·        Cotton blue: It stains the chitin on the fungal cell wall and other fungal structures

 

The stain will give a blue-colored appearance to the fungal spores and structures, such as hyphae.

 

Reagents of Lactophenol Cotton Blue (LPCB) Staining

 

A preparation of 50ml Lactophenol cotton Blue staining solution is made up of:

·        Distilled water 50ml

·        Cotton Blue (Aniline Blue) 0.125g

·        Phenol Crystals (C₆H₅O₄)  50g

·        Glycerol 100ml

·        Lactic acid (CH₃CHOH COOH) 50ml

·        70% ethanol

 

Preparation of Lactophenol Cotton Blue solution

Lactophenol Cotton Blue solution is prepared for over two days leaving the reagents undisturbed to allow dissolving and maturation.

1.     Day 1: Dissolve the cotton blue in distilled water and leave to rest overnight.

2.     Day2: Using protective gloves, add phenol crystals to lactic acid in a glass beaker and stir using a magnetic stirrer until the crystals dissolve.

3.     Add glycerol

4.     Filter the Cotton blue and the distilled water into the phenol + glycerol +lactic acid solution and mix.

5.     Store at room temperature.

 

Procedure of Lactophenol Cotton Blue (LPCB) Staining

1.     On a clean microscopic glass slide, add a drop of Lactophenol Cotton Blue Solution

2.     Add the fungal specimen to the drop of alcohol using a sterile mounter such as an inoculation needle.

3.     Tease the fungal sample using needle, to ensure the sample mixes well with the alcohol.

4.     Carefully cover the stain with a clean sterile coverslip without making air bubbles to the stain.

5.     Examine the stain microscopically at 40X, to observe for fungal spores and other fungal structures.

 

 

Results and Interpretation

Fungal spores, hyphae, and fruiting structures stain blue while the background stains pale blue.

 

          

                                     Penicillium                                 Aspergillus

Limitations

·        It can only be used as a presumptive identification method of fungi which should be followed up with other diagnostic tools such as biochemical and cultural examination.

·        The components of the solution should be used before expiry, including the use of the solution before it expires.

·        The solution may disrupt the original morphology of the fungi.

·        The stain can only be used to identify mature fungi and its structures and not the young vegetative forms of fungi.

·        The stain can not be stored for a long period of time.

 

Applications

Clinical diagnosis:

·        Identifying fungal infections in various body sites like skin, lungs, blood, and cerebrospinal fluid. 

·        Differentiating between different types of fungi based on their staining characteristics. 

·        Monitoring the effectiveness of antifungal treatments.

 

Research applications:

·        Studying fungal cell wall composition and structure 

·        Investigating fungal interactions with host tissues 

·        Analyzing fungal diversity in environmental samples 

·        Evaluating antifungal efficacy in vitro 

 

 

Common fungal stains and their characteristics:

 

·        Periodic Acid-Schiff (PAS):

Used for detection of fungi in tissue sections. Stains fungal cell walls pink, making it useful for identifying both yeast and hyphae. Oxidation of fungal polysaccharides results in the production of aldehydes that react with Schiff reagent and fungal cell walls appear pink/magenta in colour.

Procedure

1. Tissue sections are treated with periodic acid.
2. Stain with Schiff reagent.
3. Observe under bright-field microscope.

Results:

• Fungal structures appear magenta/red; tissue background lightly stained.

Applications:

• Detection of fungi in tissue sections (biopsy specimens).
• Identification of pathogenic fungi in histopathology.
• Examples: Candida albicans, Histoplasma capsulatum.

           

                                                           Candia hyphae overlying a     plaque of squamous cells- Fungal esophagitis- PAS stain

Calcofluor White:

• A fluorescent stain that binds to chitin and cellulose in fungal cell walls, allowing for rapid visualization with a fluorescence microscope. Fungi fluoresce bright blue-white under UV light.

Procedure

1. Place fungal material on a slide.
2. Add a drop of Calcofluor White.
3. Observe under fluorescence microscope.

Results:

• Fungal structures glow bright blue/white; background remains dark.

Applications:

• Rapid detection of fungi in clinical specimens (e.g., skin scrapings, sputum).
• Study of spores, hyphae, and yeast forms.
• Examples: Candida, Trichophyton.

 

Calcofluor white stained Candida albicans

showing true hyphae (*) and pseudohyphae (+).

Gomori's Methenamine Silver (GMS):

Gomori's Methenamine Silver (GMS), is a histological stain that highlights polysaccharides in tissue, primarily used to detect fungi by staining them black. It oxidises  carbohydrates in fungal cell walls to aldehydes, which then reduce silver ions to visible black metallic silver,  and is good for diagnosing fungal infections like Pneumocystis carinii pneumonia (PCP) and identifying opportunistic pathogens. GMS is considered highly sensitive for detecting fungi in tissue samples.   

 

GMS

Fungal staining methods with specific applications

Staining Method	Principle	Procedure (Brief)	Appearance / Results	Specific Applications / Examples
1. Lactophenol Cotton Blue (LPCB) Staining	Cotton Blue binds to chitin in fungal cell walls; phenol kills fungus, lactic acid preserves structure.	Mix fungal material with LCB, cover with coverslip, observe under bright-field microscope.	Hyphae, conidia, and spores appear blue; background pale.	- Identification of fungi in culture (e.g., Aspergillus, Penicillium). - Study morphology: hyphal septation, spore arrangement.
2. Calcofluor White Staining	Fluorescent dye binds to chitin and cellulose in fungal walls; fluoresces under UV light.	Mix fungal material with Calcofluor White, observe under fluorescence microscope.	Fungal structures bright blue/white; dark background.	- Rapid detection of fungi in clinical samples (e.g., skin scrapings, sputum). - Visualization of yeast and filamentous forms (e.g., Candida, Trichophyton).
3. Periodic Acid-Schiff (PAS) Staining	Periodic acid oxidizes polysaccharides to aldehydes, which react with Schiff reagent; fungal cell walls stain magenta.	Treat tissue section with periodic acid (Schiff reagent). Observe under bright-field microscope.	Fungal structures appear magenta/red; tissue lightly stained.	- Detection of fungi in tissue sections or biopsies (e.g., Candida albicans, Histoplasma capsulatum). - Diagnosis of invasive fungal infections.

In short

• LPCB → culture/fungi morphology
• Calcofluor White → rapid clinical detection, fluorescence
• PAS → tissue biopsy, invasive fungal infections