Determination of Cell Mass for Measurement of microbial Growth

 The growth results into synthesis of new cellular material. Therefore, it results into the increase in cell mass. Hence, methods used to determine cell mass can be useful to measure the growth.

  Further, every cell possesses a specific amount of cell mass. Hence, this can be also useful to find out probable number of organisms present in the growth culture.

Criteria used to determine cell mass

Every cell possesses a specifie chemical composition, dry weight as well as the volume. Hence various methods based on determination of chemical constituents of cell, volume and dry weight are applied to measure cell mass.

1]. Determination of cellular nitrogen and protein content

• Every cell possesses a fixed proportion of protein and nitrogen. To determine them, cells are first harvested, washed and then used for quantitative determination.
• Commonly micro Kjeldahl method is used to measure cellular nitrogen. This method is much laborious.
• Nitrogenous matter of the medium is also estimated together. Therefore this method is not used for routine cell mass determination.

2] Determination of cellular nucleic acid content

• Like nitrogen and protein, each bacterial cell possesses a fixed proportion of RNA and DNA. Hence their estimfuion can also be a useful criterion to measure cell mass.

3] Determination of cellular phosphorus and sulphur contents

• Phosphorus and sulfur are other major elemental constituents of cell, apart from carbon and nitrogen. They also occur in a specific proportion in cell.
•  Hence, chemical estimation of cellular phosphorus and sulfur can be useful to measure cell mass.

4] Determination of dry weight of cell

• This is one of the highly useful methods to measure growth. However, it can be used only when cell population is much dense in the culture.
• The cells are separated from culture by centrifugation or filtration, washed thoroughly and then dried in oven at 50-55°C. Then their dry weight is determined.
• However in some instances, dry weight of cells may not be indicative of living matter.
• E.g. Bacteria can accumulate large amount of poly B hydroxy butyric acid as reserve material during late log phase without causing any increase in other cellular constituents. It may account for 70% of total dry weight in some instances.

5] Determination of packed cell volume (PCV)

• Each bacterial cell belonging to a particular species has a specific volume. Increase in cell mass and cell number results in to overall increase in cell volume.
• Hence, methods determining cell volume of bacteria can be used to measure growth.
• This is achieved by centrifugation of a fixed volume of growth culture and then the settled cell mass is measured as packed cell volume.

6] Determination by turbidimetric method

• Another method of measurement of cell mass is an optical method, by determining turbidity resulting due to growth in broth cultures.
• When, a beam of light is allowed to pass through a suspension of bacteria, the bacterial particles coming in the path of light cause scattering and absorption of light depending upon the cell density as per Beer-Lambert's law.
• Therefore amount of light transmitted through the culture suspension is reduced.
• The reduction in the amount of transmitted light, therefore, depends on the cell density or cell mass in the culture suspension.
• It is measured as % transmittance or optical density (O.D.) by the use of photoelectric colorimeter or spectrophotometer or densitometer.
• Usually the O.D. is measured at 540 nm wavelength of light. This is because at this wavelength light absorption by medium will be maximum and hence it will give more correct value of O.D. of the bacterial suspension.
• Another method of measuring cell density by applying same principle is through use of Nephelometer.
• Nephelometer does not measure transmitted light, passing through the culture suspension, but it measures intensity of light scattered by bacterial particles.
• The diagrammatic presentation of measurement of cell density by both photoelectric colorimeter and nephelometer is shown in  figure below.

Diagrammatic presentation of measurement of cell density by photoelectric colorimeter.

• a] Adjustment of instrument to read zero optical density by use of uninocculated culture medium as "blank" tube. 
• b] Measurement of optical density of the growth tube, more the turbidity, greater the optical density.

Advantages and disadvantages of methods for determination of cell mass

1. Of all the methods, cell mass determination by turbidimetric method is most convenient and reliable.
2. Other methods especially involving determination of cell constituents such as cellular nitrogen, protein nucleic acids, phosphorous or sulfur has only academic value. In routine, these methods are complex, and impractical. Media constituents may interfere with estimation.
3. The methods provide information about total cell mass; both living and non-living.

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Electronic count by coulter counter

Electronic count by coulter counter

This is a highly sophisticated technique for counting bacterial cells present in the culture. The method is named so after its Inventor. 

The electronic device consists of tube having small orifice of about 10aua30 mm in diameter, which connects two compartments of the counter, which contain conductive medium. 

  The interior and exterior of the tube is connected with electrodes. The conductive medium completes the circuit.

  When, bacteria pass through the small orifice, the conductivity between two compartment drops momentarily.

  This is electronically recordcd in the counter. Thus as the bacteria pass through the orifice from one compartment to the other, each time they give electronic pulse to be counted. Thus the method involves a much rapid and accurate count.

The other modification of the technique is the use of a photodiode. Near the orifice, a photodiode and sensor are placed on the opposite sides. 

  As the organism passes through the orifice, it obstructs the path of light. This is sensed by the sensor and recorded in the counter.

Figure : Electronic coulter current counter method determining bacterial cell numbers. 

1. Flow of the growth medium through a tube is allowed through a narrow capillary tube with a small orifice. Both ends of the tube are connected to electronic circuit through electrode. 
2. Through the orifice only one cell is able to pass at a time. Passage of cell through orifice causes momentary drop in conductivity, which is gives electronic pulse, to be counted.

Limitations of electronic count method

1. However, the method has got a major limitation that it cannot distinguish between dead or living organisms as well as dust particle. Every particulate matter can give rise to pulse to be counted as the organisms. 

  2. Many times if the organisms are larger than the diameter of the orifice, they may plug into It. Similarly clumps of bacteria cannot pass through. Hence the specimen should be initially treated to separate the clumps and then used for the count.

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Microbiology of Air: Sources of Micro-organisms in Air

  Air is the mixture of gases that makes up the Earth's atmosphere. It is a mixture of about
- 78% Nitrogen,
- 21% Oxygen,
- 0.9% Argon,
- 0.04% Carbon dioxide and very small amounts of other gases.
Air also contains the variable amount of water vapours.

  Air microbiology is concerned with the study of living microbes in the air. They are usually referred to as bioaerosols. Atmosphere is actually unsuitable for growth of microorganisms due to extreme temperature variations, solar radiations, lack of nutrients, low amount of available water etc.

  Therefore, the number of microorganisms in air is less than in the other natural environments. However, there is still a large enough number that they can affect the atmosphere.

Microbes in air have an opportunity to travel long distances with the help of wind. They are ecologically significant because they can be associated with disease in humans, animals and plants.

  Most of the organisms are found in the lower region of atmosphere. Atmosphere is usually occupied by those forms of microorganisms that are resistant to adverse conditions in the air. They can be bacterial spores or cysts, capsulated bacteria, fungal spores, enveloped viruses etc.

Sources of Micro-organisms in Air

• Entry of microorganisms in air takes place through various ways.   
• Natural sources such as soil, lakes, oceans, animals and humans.
• Unnatural sources such as sewage treatment plants, animal rendering, fermentation processes and agricultural activities.
• Soil is one of the source.
• Wind blow disturbs soil surface and liberates the soil microorganisms into the air.
• These microorganisms remain suspended in air for long time.
• Manmade actions like plugging and digging.
• Plant and animal surfaces.
• Large water bodies like oceans and bays.
• Water droplets or aerosols produced by wind or tidal actions at surface of these water bodies.
•  Most droplets are produced from top 0.1 mm water surfaces called as 'micro layer'.
•  It consists of many more micro-organisms than from deep layers.
• Bubbles from surface add thousands of microbes in air.
• Most significant source is the man himself. Organisms from oral, nasal, rectal passages of man and animals come in air.
• Human activities like coughing, sneezing, laughing and even talking.
• Such biological aerosoles may spread bacteria upto distance of about 15 feet.
• Infected persons release pathogens.
• Respiratory pathogens are drought resistant and can survive in air for long period.
• Air in the hospital is a site where microbial load is always very high.
• Many human infections are caused by microorganisms from atmosphere.
• For example - Epidemics of Legionnaires disease (a severe form of pneumonia caused by bacterium Legionella) were due to aerosols generated from contaminated water of air conditioning equipments.
• Aerosols formed by high speed drills in dental clinics add a large population of bacteria.
• Various industrial, agricultural, municipal are responsible for the generation of bioareosols.
• For example - Sprinkler irrigation of crops and forest land with effluent from sewage treatment plants, trickling filter beds in sewage treatment plants, slaughter houses, spray washing processes etc.

Infectious dust, Droplets & Droplet Nuclei

(i) Infectious dust:

• Dust in air arises from sand, soil, ash, lint from bedding, clothing, carpets.
• It usually contains all saprophytic organisms.
• Dust loaded with saprophytic organisms is harmless for human. But if it is loaded with air borne pathogens, it is called as 'infectious dust.
• Air borne pathogens are added to the dust by body secretions.
• When secretions are dried , pathogens remain in dust and form infectious dust.
• Nasal or throat secretions on the handkerchief eventually dry and leave some residual material.
• During sneezing or coughing, large aerosol droplets get expelled and settle on floor or bed clothes.
• Moisture content of these droplets evaporates leaving behind residue.
• These residues are then disturbed during the handling of handkerchiefs, sweeping of floor, bed making This makes the dust infectious.
• If it is inhaled by healthy persons, they may get infected.

(ii) Droplets:

• Sneezing or coughing activity of respiratory tract infection persons expels (exhales) millions of micro droplets of saliva and mucus along with varying number of micro-organisms.
•  Such particles are called as droplets. Thus, droplets contain thousands of living micro- organisms along with saliva and mucus.
• Most of the droplets are larger in size (0.1 mm in diameter).
•  Such droplets settle rapidly, within shorter distance from their source of origin before drying.
• Even if such droplets are inhaled by a healthy person before their settling, they are immediately trapped in nasal baffles and in the nasopharynx material and are prevented from reaching upto lungs.
• Thus, droplets are not that much dangerous as far as the spread of infection is concerned.

(iii) Droplet Nuclei :

•  In warm and dry atmosphere, small droplets are dried due to evaporation of moisture content leaving behind dried mucous with live bacteria.
• They are called as 'droplet nuclei'. They are very much small in size with diameter less than 0.1 mm.
• Such droplet nuclei can remain in air for longer period of time (upto hours to days).
•  The dried mucus protects the organisms present at the center.
• They are also carried away to a longer distance.
• If these are inhaled by health persons, they directly enter into the lungs, without capturing in mechanical traps of nasal baffles and mucus of nasopharynx.
•  The organisms from droplet nuclei directly settle in the alveoli of lungs.
• Chances of spread of infection via droplet nuclei increases when people are crowded together.
• Frequency of air borne infections is more in winter when people prefer to live in crowd places.

   In modem days, the chances of spreading infection are increased because of modern urban areas like buses, aero planes, restaurants and bars which are always crowded.
 The diseases caused by Klebsiella pneumoniae, Streptococcus pneumoniae and Mycobacterium tuberculosis may spread through droplet nuclei.

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Sampling Methods for Microbiological Examination of Air

Sampling Methods for Microbiological Examination of Air

Microorganisms in air cause a problem of contamination in food industries, milk processing plants, industrial processes, pathological and research laboratories and mainly in operation theatres.

   Therefore, measurement of no. of organisms in air at such places is important. Microorganisms in air may be present in free form or in the form of clumps or chains.

  They may adhere to the dust particles or to saliva or to mucus of man. Several methods have been developed for the microbiological examination of air.

  Simplest method is exposure of sterile nutrient medium plates to air for different time periods The plates are then incubated during which the settled organisms grow and form colonies.

  The number of organisms can be calculated from the number of colonies. But this gives a rough idea about the numbers and kinds of microorganisms in air. Because the volume of air coming in contact with plate is not known.

  Therefore, the advanced techniques like solid impaction, liquid impingement, filtration, sedimentation, centrifugation, electrostatic precipitation, thermal precipitation etc. are used. Among these, solid impaction and liquid impingement techniques are most commonly used.

1. Solid Impaction (Sieve Device) :

• Number of sampling devices which work on the principle of impaction of airborne particles.
• In these, an aspirator sucks a known volume of air which collides with nutrient agar in plate.
• This causes the microbes in the air to stick to the surface of an agar medium.
• The plates are then incubated at specific temperature for a specific time to form the colonies.
• These are the most often used methods of detection and enumeration of microbes in air.
• The most widely known device that is based on the solid impaction is the Sieve Device.
• The sieve device is of two  types : Single Stage, Multistage Device

a). Single Stage Device :

• It is mechanically very simple.
• It consists of a metal box with a metal cover.
• The cover has a large number of evenly spaced small holes.
• Under this a petriplate containing agar medium can be kept.
• A measured volume of air is drawn through these hole. The particles containing microorganisms are allowed to settle on the agar surface.
• Plates are then incubated for specific time. Then microbial colonies on agar surface are counted.

Andersen's Air sampler & Single Stage Sieve Device

b). Multistage Device :

• It consists of perforated plates with progressively smaller holes at each stage.
• Allows particles to be separated according to size.
• Best known multi-stage samplers is Andersen's air sampler
• In this, air is drawn through a series of six circular plates each perforated with 400 holes.
• The plates in series have progressively smaller holes.
• Therefore, the largest particles being deposited in the first while the smallest in last petri dish.
• Viable particles can be collected on a variety of bacteriological agar media.
• Incubated for counting and identification.
• All the particles collected are sized aerodynamically and can be directly related to human lung deposition.

Advantages of Sieve Device

1. The volume of air to be analyzed can be measured.
2. An air is drawn with high speed. Therefore, the particles of smaller size that might be escaped can also be settled on agar surface.
3. There will be even distribution of air sample on agar surfaces that results in formation of isolated colonies.
4. This is helpful to take a colony count.
5. The methods are very simple and efficient.
6. Therefore, they are most widely used.

2. Liquid Impingement Device :

• In this, a known volume of air is passed in a sterile broth medium, where micro-organisms from air are trapped.
• The aliquots of broth are then used for plating.
• The plates are incubated at room temperature for specific time and the colony count is taken to determine the number of micro-organism.
• There are several devices, among which the most important one is Bead Bubbler Device.

Bead Bubbler Device

• It consists of 250 ml capacity suction flask with side arm linked to suction pump and flow meter.
• The flask is filled with sterile nutrient broth.
• The sterile glass beads of size 5 mm in diameter are also placed in the flask.
• A glass bubbler is fitted in the flask with rubber stopper.
• Diameter of glass bubbler is approximately 14 mm.
• It has many small openings at the base with3 mm diameter.
• A suction flask is added with sterile nutrient broth medium along with sterile glass beads.

 

• The bubbler is fitted with rubber stopper.
• With help of suction pump and flow meter, known volume of air is allowed to pass through medium.
• Air enters into broth medium in the form of bubbles.
• Because of glass beads bubbles are formed and the clumps or chains of organisms are broken down.
• Aliquots of medium are used to culture on solid agar medium & plates are incubated at room temperature.
• Colony count is taken & number of organisms is determined.

Advantages

1. Volume of air to be analyzed can be measured
2. Because of glass beads and bubble formed, the micro-organisms from the clumps and chains are separated from each other.
3. Thus exact count can be drawn.

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