Demonstration of bacterial motility by hanging drop method

Hanging drop method is useful in determining the size, shape and movement of living microorganisms, especially bacteria without staining them to see their motility due to flagella.  Bacteria, because of their small size and a refractive index that closely approximates that of water, do not lend themselves readily to microscopic examination in a living, unstained state.  However, hanging drop method is useful to: 1) Observe cell activities such as motility and binary fission. 2) Observe the natural sizes and shapes of the cells, since heat fixation and exposure to chemicals during staining cause some degree of distortion.

Aim

To determine the motility of bacteria by hanging drop method

Principle

Bacteria show two types of motility. One is true (actual) motility and the other is Brownian movement.  True motility is due to flagella where as Brownian movement is a vibratory movement of the cells due to their bombardment by water molecules in the suspension.  To determine whether a given organism is motile or not, it is essential to differentiate between true motility and Brownian movement.   Hanging drop preparations make the movement of microorganisms easier to see because they slow down the movement of water molecules in the closed environment of cavity slide.

Requirements

24 hr broth culture of Bacillus and Streptococcus spp.

Bunsen burner, Inoculating loop, Cavity slides, Coverslips, Petroleum jelly, Cotton swabs, Microscope etc.

Procedure

1.      With a cotton swab, apply petroleum jelly on the four corners of a clean coverslip.

2.      Using sterile techniques, a loopful of the culture is placed on the centre of the coverslip.

3.      Place the depression slide, with the concave surface facing down, over the coverslip so that the depression covers the drop of culture.  Press the slide gently to the coverslip.

4.      Turn the slide right side up so that the drop continues to adhere to the inner surface of the coverslip.

5.      Focus the drop edge under the low power objective (10X) and reduce light source by adjusting the Abb’e condenser. Repeat using high power objective (40X).

Observations

Rod shaped cells of Bacillus sp. moved swiftly across the microscope whereas round shaped cells of Streptococcus sp. showed no motility.

Result                                                                                                                                                       

In the given cultures, Bacillus sp. is having true motility and Streptococcus sp. is non motile.

B Cell Receptor and Antiboby Production

Anything that is non-self or foreign to the body is considered as  antigens.  Once antigens enter our body, lymphocytes produce specific immunological reactions against the antigens in order to remove them and to prevent the development of  an infection or disease due to that particular antigen. This immune response can be humoral or cell mediated. Humoral immune response results in the production of antibodies (Ab) by B lymphocytes. Cell-mediated immunity does not involve antibodies. Rather, it involves antigen-specific cytotoxic T-lymphocytes, the activation of phagocytes and the release of various cytokines in response to antigen.

Antigens (An) are usually  bacteria, bacterial products, fungi, viruses or other parasites.  Entire organisms  usually do not function as the antigen. Particular macromolecules of these infectious agents usually act as antigens. Proteins are the major antigens and polysaccharides coming next to it. Lipids and nucleic acids of these microorganisms do not act as antigens unless they are coupled with proteins or polysaccharides.  Antigens are also called antibody generators as they induce the immune system to produce antibodies. Hence, Antibodies are glycoproteins produced in response to  antigens by the host immune system whose function is to eliminate the entered antigen. 

In order to produce antibodies, first the antigen should activate the B lymphocytes. Activated B cells are called Plasma cells or Plasma B cells.  Plasma cells are white blood cells that could secrete large volumes of antibodies or immunoglobulins. These secreted immunoglobulins are transported by blood plasma and lymphatic system to destroy the entered antigens. 

The B cells, a type of white blood cells, are produced by multipotent hematopoietic stem cells (HSCs) in the bone marrow.  Mature B-cells that have not been exposed to antigens are called naive B cells.

Naive B cells carry special receptors on their cell membrane to which the An binds. These receptors are called B Cell Receptors or BCRs. BCRs are made up of two parts. First part is the transmembrane antibody attached to the cell membrane of the B cell, to which the  antigen binds. Antibodies of  any one isotype like IgD, IgM, IgA, IgG, or IgE are acting as BCRs.  These antibodies are also called membrane immunoglobulin (mIg). This region is oriented outward, away from the cell. The second part is the signal transduction transmembrane heterodimer proteins embedded in the cell membrane of B cells called Igα & Igβ. These heterodimers are held together by disulfide bonds. The transmembrane immunoglobulin and the heterodimer proteins together make up the so called BCRs. 

Each individual B cell has around 50,000 BCRs on its surface. But one B cell carries BCRs specific for only one particular antigen. Thus, the B cell population of an adult individual carries BCRs for as many as 10 ¹³    different antigens. Means 10 ¹³   different and undifferentiated B cells are present in an adult human body. They circulate in blood, awaiting the activation by specific antigen. 

When an An is captured, the membrane receptor communicates with the nucleus through the signal transduction heterodimer proteins and B Cell is activated. Upon activation,  these B-cells immediately undergo clonal proliferation and differentiate into antibody producing plasma cells (effector B cells) and memory cells.  Plasma cells will secrete soluble antibodies and these Abs circulate through the bloodstream to identify the antigen that had induced its synthesis.  Such circulating antibodies are also present in the serum, tissue fluid and mucosal surfaces of vertebrates. Memory cells remain in circulation for very longer periods and produce secondary response. When the same An enters the body for a second time, memory cells  proliferate and differentiate into plasma cells, which then clear the antigen.

                                                    Image Courtesy: immunology.org

Sources of Microorganisms in Air

Aero microbiology is the study of intramural or indoor and extramural or outdoor microorganisms in air.  In other words, aero microbiology deals with the distribution, transmission and existence of microorganisms like bacteria, fungi, viruses, yeast and protozoans in air. Air is not a natural environment for microorganisms.  The physical and chemical parameters prevailing in air do not support the growth and reproduction of microorganisms. Microorganisms in air are exposed to sunlight, UV radiation, desiccation, less nutrients etc which will affect the existence of microorganisms. Hence the amount of microorganisms in air is less than soil and water.  

Vertical distribution of microorganisms in air is controlled by air currents, wind flow etc. whereas their horizontal distribution is affected by various physical and chemical factors.  The distribution of microorganisms considerably reduces as the altitude increases. At Higher altitudes, microorganisms are exposed to decrease in temperature, less oxygen content, low atmospheric pressure, low water availability, less organic carbon etc. which will limit their distribution. These conditions limit the distribution of microorganisms above the troposphere layer of Earth. Only resistant spores are found above the troposphere layer, but in low concentrations. 

Microorganisms present in air are liberated from various other sources. These various sources include soil, water, plant and animal surfaces and human beings.  Microorganisms remain in the air for a varying time period depending upon speed of air currents, size of particles to which they are attached and humidity of the atmosphere. In still air microbes settle easily whereas a gentle air current can keep the microbes suspended indefinitely in air. Also organisms attached to dust particles or droplets settle out faster than the free organisms which are only slightly heavier than the air. A humid atmosphere contains less amount of organisms than a dry one as organisms are carried down by the droplets of moisture.  This explains why the microbial load is more in summer than in winter.

 Sources of microorganisms in air include

1.      Soil

2.      Water

3.      Plant and animal surfaces

4.      Human beings

1.   Soil

Soil is the most common source of microorganisms in air. From soil microorganisms are liberated to air by various environmental activities as well as by human activities. Environmental activities like wind blow, air currents liberate soil microorganisms into air and these organisms will remain in the air suspended for longer periods. Human activities like digging, ploughing etc. will also liberate microorganisms from soil to air. An active soil environment liberates more microorganisms to air than less active soil environment.  Also air above rich, fertile and cultivated soil shows a higher viable count than sandy and uncultivated soil.  Similarly, soil covered with vegetation liberates less amount of microorganisms into air than bare surfaces as bare surfaces can be easily acted upon by wind and air currents.

2.  Water

Microorganisms are liberated from water into air as droplets or aerosols.  Here also environmental activities like splashing of water by wind and tidal action as well as human activities like swimming, water sports etc liberate microorganisms into air. 

3.  Plant and Animal Surfaces

Microorganisms on plant and animal surfaces are liberated to air. These organisms can be commensals or pathogens.   70% of plant diseases are transmitted through air. Plant pathogens spread over long distances. Eg: Spores of Puccinia graminis.  Animal diseases are less frequently transmitted through air.

4. Human beings

Human beings are the main source of microorganisms in the air. Surface flora of the human body is shed at intervals. In addition to that human beings also produce bioaerosols which may contain commensals as well as pathogenic microflora of mouth and upper respiratory tract by the activities like coughing, sneezing, talking, laughing, singing etc. Particles suspended in air are called aerosols. Bioaerosols contain biological contaminants like pathogenic bacteria, virus, microbial toxins etc. which on ingestion or inhalation cause infectious diseases in human beings.  Bioaerosols vary considerably in their size and composition. Their size ranges between 0.02  to    100 μm.  Based on size bioaerosols are divided into droplets, droplet nuclei and infectious dust. Composition of bioaerosols depends on the type of microorganisms or toxins they are attached with and also the type of particles they are attached to like mucus, dust etc. 

4.1. Droplets

Droplets are formed by human activities like coughing, sneezing, talking, laughing etc and also during disease diagnosis procedures like suctioning and bronchoscopy. Droplets consist of saliva or mucus, epithelial cells, cells of the immune system and various microorganisms. Hundreds of microorganisms can be seen in such droplets and these organisms can be pathogenic, if discharged from infected persons.  Usually pathogens of the respiratory tract are liberated as droplets. The size of the droplet determines its period of suspension in air.  Droplets are usually having a larger size, greater than 10μm or more and hence, they will settle rapidly in still air.True aerosolization does not occur in the case of droplets. They travel less than 1m through the air and are immediately deposited on the nasal or oral mucosa of the new host or in their immediate environment. If they are inhaled, they are usually trapped on the moist surfaces of the respiratory tract and cause the upper respiratory tract infections. They cannot move to the lower parts of the respiratory tract because of their size and hence cannot cause lower respiratory tract infections.

4.2. Droplet nuclei

Droplet nuclei are airborne particles originating from droplets by the evaporation of large droplets. Droplets in a warm and dry atmosphere evaporate rapidly and the solid material left after drying up of the droplet is called droplet nuclei. They are less than 5 μm in size (usually between 1-4μm) and contain microorganisms, dust particles, skin cells and other debris. Here aerosolization takes place and droplet nuclei remain suspended in the air for longer periods of time. Droplet nuclei are widely dispersed by air currents, remain for hours or days and are inhaled by susceptible hosts. Once inhaled, they can escape the mechanical traps of the upper respiratory tract and enter the lungs to cause lower respiratory tract infections.  They are more potential agents of infection than droplets and play an important role in  transmission of airborne diseases, particularly respiratory infections. The role of droplet nuclei in transmission of  airborne diseases was first studied by Wells in 1955. 

To cause an infection, microorganisms present in droplet nuclei should be viable.  Viability of organisms in droplet nuclei is determined by atmospheric conditions like humidity, sunlight, temperature, also by the size of particles bearing the organisms and degree of susceptibility or resistance of microbial species to the new physical environment. 

4.3. Infectious dust

Large droplets settle out rapidly from air onto various surfaces including cloths, floor, wall, table tops and other exposed surfaces and get dried. These droplets may include nasal and throat discharges of patients containing infectious pathogens.   Disturbance of these dried materials during bed making, sweeping of the floor, handling of contaminated handkerchief etc. will liberate infectious dust into the air. These infectious dust remain suspended in air for longer periods of time and are a serious hazard in hospital areas. Also laboratory practices like opening of frozen bacterial cultures or the cotton plugs dried after being wetted by the culture broth will liberate infectious dust in laboratories. 

View my Video: " Sources of Microorganisms in Air"

Youtube Link: https://www.youtube.com/watch?v=dlzdUd_yLq0&feature=youtu.be

Gram Staining Procedure

Gram stain was introduced in 1880 by the Danish bacteriologist Christain Gram.  Based on this staining reaction almost all bacteria can be separated into two large groups called Gram positive and Gram negative.

 

Aim

To differentiate between two principal groups of bacteria: gram positive and gram negative.

 

Principle  

It is a type of differential staining which uses four reagents and has got four steps. The heat fixed smear of the organism is first treated with the primary stain called Crystal violet.  It is a basic dye and its function is to impart its color to all cells.  At this stage all the organisms appear in violet color.  In the second step, after washing, the smear is treated with Gram’s Iodine.  This reagent acts as the killing agent as well as the mordant.  Mordant is a substance that increases the cell's affinity for a particular stain.  It binds with the primary stain and forms an insoluble crystal violet- iodine (CV-I) complex.  All cells appear purple black at this stage.  In the third step, the smear is treated with the decolorizing agent, absolute ethanol.  During this process, some bacteria loose the CV-I complex, whereas some retain the same.  In the final step, the smear is treated with the counter stain or secondary stain called Safranin.  The organisms which have lost the CV-I complex take up this red dye and appear red in color and are called Gram negative bacteria.  The organisms which did not lose CV-I complex will not take up the secondary stain and remain violet in color. They are called Gram positive bacteria.

 

The difference in the chemical and physical nature of the bacterial cell wall is responsible for this difference in response to gram stain.  The gram negative cell wall is thin, complex, multilayered and contains relatively high lipid contents, in addition to proteins and mucopeptides.  The dehydrating agent (absolute alcohol) readily dissolves the higher amount of lipids leading to the formation of large pores in the cell wall which do not close appreciably on the dehydration of the cell wall proteins.  Through these pores the CV-I complex leakage takes place and the cells take up the counter stain.  In contrast, the gram positive cell walls are thick and chemically simple, composed mainly of protein and cross-linked polypeptides.  When treated with alcohol, it causes dehydration and closure of cell wall pores, thereby preventing the loss of CV-I complex and cells remain violet colored.

 

Requirements

 

24 hr old cultures of Escherichia coli and Staphylococcus spp.

 

Gram staining reagents:

   Crystal violet

   Gram’s iodine solution

   95 % ethyl alcohol

   Safranin          

 

Wash bottle of distilled water, staining tray, droppers, inoculating loop, glass slides, blotting paper, Bunsen burner, microscope etc.

 

Procedure

 

·         On separate slides, make thin smears of the cultures provided, air dry and heat fix

·         Flood the smear with Crystal violet for 30 sec

·         Wash the slides with distilled water for few seconds

·         Cover each smear with Gram’s iodine solution for 30 sec

·         Decolorize the smears by adding ethyl alcohol drop by drop, until no more color flows from the smear

·         Wash the slides with distilled water and drain

·         Apply Safranin to smears for 30 sec

·         Wash with distilled water, blot dry with blotting paper and air dry

·         Observe under oil immersion objective (100X).

 

Observations

Escherichia coli- Rod shaped (bacilli) cells in chains and appeared red in color

Staphylococcus - Round shaped (cocci) cells in grape like clusters and appeared violet in color.

 

Result                                                                                                                                        In the given cultures, Staphylococcus spp. belongs to Gram positive bacteria and E. coli to Gram negative bacteria.              

 

Pure Culture preservation strategies

1.  Introduction.

Maintenance of pure cultures of bacteria requires frequent attention so that the organisms are not lost or contaminated. They have to be transferred at regular intervals to suitable media before they are destroyed by their own waste products. But repeated sub culturing will lead to the loss of biological, immunological and cultural characteristics of the organism. Usually, more than 5 subcultures are not preferred.

In microbiology laboratories we maintain culture of different strains of microorganisms as they are required for laboratory classes, research works, or for other particular procedures. They are called Stock culture collection. Microbial cultures are also maintained as reference strains for taxonomic studies. American Type Culture Collection (ATCC) in USA maintains 1 million ampules of different strains of microorganisms.  Microbial Type Culture Collection & Gene Bank (MTCC) in Chandigarh is another central collection unit in India.

Preservation is the maintenance of microorganisms for extended period of time in viable condition without contamination or lose of genetic and morphological characteristics. Preservation methods either stop or slow down the metabolism and multiplication of microbes. Different methods are used to maintain the pure culture of microorganisms. However, no single method is complete for preserving all types of microorganisms.

2.                       Types of culture preservation methods

Based on the period of storage, culture preservation is divided into Short term methods and Long term methods.

Short term methods	Long term methods
·                        Agar slant cultures (Sub culturing)  & Refrigeration	Mineral Oil or Liquid Paraffin Method
	Saline suspension storage
	Drying in Vacuum
	Storage at low temperatures (Cryopreservation)
	Lyophilization (Freeze drying)

3.                       Short term methods (1 Type)

3.1.                 Agar slant cultures (Sub-culturing) and Refrigeration

Pure culture can be maintained by periodic aseptic sub-culturing on to the suitable media. Type of media, storage temperature and interval between sub-culturing depends on the bacterial species. These are referred as stock cultures. Solid media is usually preferred as the toxic material produced by organisms diffuse into slant and thus away from microbial growth. Most heterotrophs can be easily maintained on nutrient agar. Nutrient agar slants in screw capped bottles are preferred as it prevent drying of the media. Disadvantage is that repeated sub-culturing leads to mutational changes in the strain.

After preparation of stock cultures, they are incubated for 24 hrs or more to get sufficient growth and stored in cool dry place or refrigerator till use. The temperature inside the refrigerator is usually 4^o C which can slow down the growth and metabolism of microorganisms. Thus nutrient use from media is considerably reduced which is useful in the maintenance of organisms. Here also periodic sub-culturing is needed as waste product accumulation adversely affect the microbial growth.

4.                       Long term methods (5 Types)

4.1.                 Mineral Oil or Liquid Paraffin Method

Agar slants in screw capped tubes are inoculated and incubated till good growth is obtained. Then it is covered with sterile mineral oil or liquid paraffin to a depth of 1 cm above the slant. This method can be used to preserve bacteria and fungi in viable condition at room temperature from months to years. Paraffin layer prevents the dehydration of the medium and also produce anaerobic condition so that microorganisms remain in a dormant state.

This method has many advantages.

The unique advantage of the method is that sub-culturing can be done without affecting the stock culture. Sub-culturing is done by taking loopful of growth under the oil from these slants, touching the loop to the glass surface of tube to drain off excess oil and then inoculating to fresh medium.

• Many species remain viable for longer periods under oil than in tubes without oil.
• Sub-culturing can be done as and when required without affecting the stock cultures.
• Useful for strains susceptible to mutations on repeated sub-culturing
• Simple and economical method as equipment like dessicator, vaccum pump etc are not needed.

4.2.                Saline suspension storage     

Sodium chloride in high concentration is inhibitory to the microbes. But 1% salt solution is useful for culture preservation and is considered as sub lethal concentration. In this method bacteria are suspended in salt solutions in screw capped tubes to avoid evaporation. Tubes can be stored at room temperature and when required, can be sub-cultured to agar slants. This is an easy method for storing bacterial cultures for 2-3 years and particularly useful in labs with limited equipment facility. 

4.3.                  Drying in Vacuum

Organisms are found to survive longer if they are dried in a desiccator than when air dried. Different methods are employed for drying in vacuum.  Organisms are suspended in liquid media and dried in a desiccator in vacuum over dehydrating agents such as H₂SO₄ or P₂O₅ (Phosphorus pentoxide). It is called Liquid drying or L –Drying. This method is particularly useful for microbes which are sensitive to freeze drying as this method involves vacuum-drying of samples from the liquid state without freezing. . The microbial suspensions can be also dried on sterile coverslips, filter paper, in small test-tubes, or more conveniently in sterile ampoules which can subsequently be sealed off in vacuo.

4.4.                 Freezing and Cryopreservation

The organisms suspended in nutrient broth containing 15% glycerol can be frozen and can be stored at -29^o C for up to 2 years.    The availability of liquid nitrogen provided another useful means for preserving stock culture. In this method dense cell suspensions are prepared in the medium containing cryopreservative agents like glycerol or Dimethyl sulfoxide (DMSO). These agents prevent ice crystal formation during freezing process which may damage microbial cells. This suspension is sealed into small ampoules or vials and then frozen at controlled rate to -150 ^o C. Then the ampoules or vials stored in a liquid nitrogen refrigerator either by immersion in a liquid nitrogen (-196°C) or by storage in a gas phase above liquid nitrogen (-150 ^o C). This method is useful for preservation of organisms that cannot withstand freeze drying.  Cultures can be maintained for 10-30 years or more without any change in the characteristics of microbes. However, this method is expensive as liquid nitrogen has to be replenished at regular intervals to replace the loss due to evaporation.

4.5.                  Lyophilisation (Freeze drying)

This method is mainly used to preserve organisms that would be killed by ordinary drying process. In this method, dense cell suspension is prepared in small vials and frozen at -60 to      - 78^o C. Vials are connected to high vacuum so that ice present in frozen culture undergo sublimation under vacuum. Sublimation is evaporation from solid to gaseous stage without passing through liquid phase. This causes dehydration of bacteria with minimum damage to delicate cell structure.

Vials are then sealed under vaccum and stored in refrigerator. Many species of bacteria can be preserved by this method for more than 30 years in viable condition without any change in characteristics. Minimal storage place is required for lyophilised cultures, 100s of cultures can be stored in a small area. Also these vial are convenient to mail in sealed containers to other laboratories. Lyophilised cultures are revived by rehydrating the opened vials by adding liquid medium and then transferring it to the suitable media. 

 Watch my VIDEO on Pure Culture Preservation Strategies