Isolation of airborne microbes by settle plate method

Principle:

The concentration and quality of micro organisms in atmosphere can have a direct bearing on human health and environment. In recent years air microbiology has gained a lot of attention. Microbiological quality of air can be considered as a mirror of hygienic conditions of any place.

Air samples can be collected in two ways: by active air samplers or by passive air sampling (the settle plates).Passive air sampling is performed using settle plates. Petri dishes containing solid nutrient medium or PDA medium are left open to air for a given period of time. Microbes carried by inert particles fall onto the surface of the nutrient, with an average deposition rate. After incubation they grow into colonies in a number proportional to the level of microbial contamination of the air. Despite a few limitations, the settle plate method is still widely used as a simple and inexpensive way to qualitatively assess the environments over prolonged exposure times. They are sterile, economical and readily available. Many sites can be checked at the same time.

Materials Required:

Petri dish, Nutrient agar, Potato Dextrose Agar, Incubator, colony counter

Procedure:

1. Prepared sterilized pert plates of  Nutrient agar and PDA

2. The plates were transported to the sites or area by using sealed bags.

3. The plates were labeled with sample number and site number.

4. Petri dishes left open to the air according to the 1/1/1 scheme (for 1 hr, 1 m from the floor, at least 1 m away from the walls or any obstacles).

5. After the exposure the plates were covered with lids and taken to the laboratory in sealed plastic bags and incubated at 37^oC for 24 hours.

6. The culture plates that showed discrete macroscopic colonies were counted using plate colony counter.

7. The concentration of airborne bacteria /fungi was expressed as colony forming units per cubic meter (cfu/m3).

Students of Microbiology in IAAM conference

• Microbiology students participated in IAAM (Indian Association of Applied Microbiologists) Conference 2017 at Vivekanada College, Tiruchengode.

• Participants from BSc  Second Year & MSc  first year, they presented the posters in conference.

Blood collection method

Blood collection method demonstrated  by Mr. E. Kumaran, faculty,dept . of microbiology, Dr.N.G.P. arts and science college.

Food Waste Disposal Methods

            

            Food waste is an untapped energy source that mostly ends up rotting in landfills, thereby releasing greenhouse gases into the atmosphere. Food waste includes organic wastes generated in hotels, restaurants, canteens, cafeterias, shopping malls and industrial parks in the form of leftover food, vegetable refuse, stale cooked and uncooked food, meat, teabags, napkins, extracted tea powder, milk products etc. It is difficult to treat or recycle food waste since it contains high levels of sodium salt and moisture, and is mixed with other waste during collection. 

Food waste can be recycled by two main pathways:

·         Composting: A treatment that breaks down biodegradable waste by naturally occurring micro-organisms with oxygen, in an enclosed vessel or tunnel or pit

·         Anaerobic digestion or biogas technology: A treatment that breaks down biodegradable waste in the absence of oxygen, producing a renewable energy (biogas) that can be used to generate electricity and heat.

Composting

​​Composting provides an alternative to landfill disposal of food waste, however it requires large areas of land, produces volatile organic compounds and consumes energy. Compost is organic material that can be used as a soil amendment or as a medium to grow plants. Mature compost is a stable material with a content called humus that is dark brown or black and has a soil-like, earthy smell. It is created by: combining organic wastes (e.g., yard trimmings, food wastes, manures) in proper ratios into piles, rows, or vessels; adding bulking agents (e.g., wood chips) as necessary to accelerate the breakdown of organic materials; and allowing the finished material to fully stabilize and mature through a curing process. 

Anaerobic Digestion

Anaerobic digestion (AD) is a microbial decomposition of organic matter into methane, carbon dioxide, inorganic nutrients and compost in oxygen depleted environment and presence of the hydrogen gas. This process, also known as bio-methanogenesis, occurs naturally in wetlands, rice fields, intestines of animals, manures and aquatic sediments, and is responsible for the carbon cycle in the ecosystems. Natural and anthropogenic sources account for 30 and 70 %, respectively, of the total methane released in the atmosphere every year. Major natural sources of methane are the wetlands and animal guts (mainly insects and ruminants) while the main anthropogenic sources have been identified in the fossil fuel processing industries, rice fields and landfills. Biological activity has been identified to be the cause for more than 80% of the flux of the atmospheric methane (Palmisano et al. 1996). 
  

Of the different types of organic wastes available, food waste holds the highest potential in terms of economic exploitation as it contains high amount of carbon and can be efficiently converted into biogas and organic fertilizer. Food waste can either be utilized as a single substrate in a biogas plant, or can be co-digested with organic wastes like cow manure, poultry litter, sewage, crop residues, abattoir wastes etc. 

Source: https://www.ecomena.org/food-waste-disposal/

WTO and Food Safety

WTO (World Trade Organization):

 -WTO is an intergovernmental organization, regulates the trade between the countries.

- It is officially commenced on 1^st  January 1995 under the Marrakesh Agreement, signed by 123 nations on 15 April 1994, replacing the General Agreement on Tariffs and Trade (GATT), which commenced in 1948.

- It is the largest international economic organization in the world.

- The WTO deals with regulation of trade in goods, services and intellectual property between participating countries by providing a framework for negotiating trade agreements and a dispute resolution process aimed at enforcing participants' adherence to WTO agreements, which are signed by representatives of member governments.

WTO agreements and Codex alimentarius

         Codex Alimentarius Commission is an organisation that develops independent science based international food standards.

1.      Agreement on Technical Barriers to Trade (TBT) is an agreement that refers to technical regulations and conformation of assessment procedures which apply to all commodities including food. The TBT Agreement also covers measures that are intended to protect human safety and health.

2.      Agreement on Sanitary and Phytosanitary Measures (SPS) this agreement covers food safety where members are entitled to establish sanitary and phytosanitary measures for the protection of human health, provided the measures are consistent with the provisions of the agreement. A common example of regulations whose objective is to protect human health is the labelling of cigarettes indicating that they are harmful to health. Sanitary and phytosanitary measures must be based and applied in a manner that would not be used as a disguised restriction that will affect international trade.

         The SPS Agreement states that all members can base their sanitary and phytosanitary measures on the international standards, guidelines or recommendations. They also have the option to make their SPS measures stricter that international standards if there is a scientific justification for it. To ensure transparency, Member countries are required to notify if they have made any changes to the SPS measures especially if it affects trade. Members have to also set up an Enquiry Point from where they can respond to requests for information on their SPS measures.

      Member countries of the WTO have the sovereign right to have a level of protection for foods that they consider appropriate as acceptable levels of risk. However, these levels of protection on foods must be based on a risk assessment.  All those Members whose measures are in keeping with international standards are within their WTO obligations. If they have made their measures more stringent than the international standard then they could be challenged to justify such measures if they lead to a trade barrier.

    These protection measures will be equal for domestic and imported products so there is no discrimination against foreign sources of food supply. Members are expected to promote the review and development of international standards. They need to accept the SPS measures of other member countries as equal even if they different from their own measures if the measures of other countries provide appropriate level of protection.

SPS Measures in the area of food safety may include

§  Control and inspection procedures

§  Pesticide tolerances

§  Food additive approval processes    

       For food safety, the SPS Agreement makes specific reference to the standards, guidelines and recommendations established by the Codex Alimentarius Commission. Members are required to also participate in meetings, within the limits of their resources, in relevant international organizations and their subsidiary bodies especially in the Codex Alimentarius Commission in the field of food safety. This helps to promote the development and periodic review of standards, guidelines and recommendations. For food safety, the SPS Agreement, will use Codex standards in the following five areas

§  Food additives

§  Veterinary drugs and pesticide residue

§  Contaminants

§  Methods of analysis and sampling

§  Codes and guidelines of hygienic practices

What the TBT Agreement contains

The TBT Agreement aims to ensure that technical regulations and standards for packaging, marking and labelling requirements, and analytical procedures for assessing conformity do not create unnecessary obstacles to trade. For Codex purposes, most of the provisions under the TBT Agreement seek to protect consumers by providing information, mainly in labelling requirements, nutritional requirements, quality provisions, and methods of analysis to promote fair trade practices.

All food standards not covered by the SPS Agreement should be considered under TBT related measures. These provisions include classification and definition, essential composition and quality factors, packaging requirements and measurements (size, weight, etc.), so as to prevent any fraudulent practices that can deceive consumers. Under the TBT Agreement governments may decide that certain international standards are not appropriate for such reasons like fundamental technological problems or geographical factors.

Sources: http://foodsafetyhelpline.com, www.wikipedia.com

Methanogens

- Methane producing microbes are called Methanogens.
- Methane is the gas and metabolic byproduct of the organisms.
- Methanogens belongs to the archaea domain, they are obligate anaerobes.
- They are very sensitive to oxygen.
- They are commonly present in wetlands and digestive tracts of animals (ruminants, humans)

- Methanogens are cocci (spherical) or bacilli (rod) shaped bacteria. 
- They are chemoautotrophic organisms ( using inorganic energy source such as molecular hydrogen, hydrogen sulphide, elemental sulfur etc.) 

- They reduce the carbon dioxide to give methane (marsh gas) by using hydrogen as electron source: 

                     CO₂ + 4 H₂ →  CH₄ + 2H₂O

- Formation of methane from microbes called methanogenesis. 

 Examples of Methanogens :

-Methanobacterium 
-Methanobrevibacter
-Methanosarcinae
-Methanococcus
-Methanospirillum
-Methaothermobacter
-Methanothrix

Metabolic Pathway of Methanogens

Source: D Browne, Patrick & Cadillo-Quiroz, Hinsby. (2013). Contribution of Transcriptomics to Systems-Level Understanding of Methanogenic Archaea. Archaea (Vancouver, B.C.). 2013. 586369. 10.1155/2013/586369. 

Overview of the three major known methanogenic pathways in Archaea. Color coding indicates the steps common to all three types (black), unique to the methylotrophic pathway (green), unique to the hydrogenotrophic (or CO2 reducing) pathway (blue), unique to the aceticlastic pathway (red), and shared between hydrogenotrophic and aceticlastic methanogenesis (purple). 2e− represents reducing equivalents, produced or consumed during each reaction. MFR: methanofuran; H4MPT: tetrahydromethanopterin; CoM-SH: coenzyme M; CoB-SH: coenzyme B; CoA-SH: coenzyme A; CoM-S-S-CoB: heterodisulfide of coenzyme M and coenzyme B; ATP: adenosine triphosphate; R: ligand bound to methylated compound that serves as substrate for methylotrophic methanogenesis. *Tetrahydrosarcinapterin is a functional analogue of H4MPT found in the Methanosarcinales order of methanogens.

- Some times the marine methanogens produce methane form acetic acid. 

Measurement of Microbial Growth by Total Viable Count Method

Objective:

To find the generation time of microorganisms by total viable count method.

Principle:

The number of cells capable of dividing and forming the colonies on solid medium are called viable number cells. Total viable count method is more convenience because there is virtually no other reliable and virtual way to count living organisms. In this method, serial dilutions of a sample containing viable microorganisms are plated onto a suitable growth medium. The suspension is either spread onto the surface of agar plates (spread plate method), or is mixed with molten agar, poured into plates, and allowed to solidify (pour plate method). The plates are then incubated under conditions that permit microbial reproduction so that colonies develop that can be seen without the aid of a microscope. It is assumed that each bacterial colony arises from an individual cell that has undergone cell division. Therefore, by counting the number of colonies and accounting for the dilution factor, the number of bacteria in the original sample can be determined.

Materials required:

-Nutrient agar

- 10- 12 hrs old broth culture

-Sterile pipette  

-L –rod

-Turn table

-Petri dishes

Procedure:

-  The bacterial sample is serially diluting upto 10⁴ dilutions (add 0.5 ml of sample in 4.5 ml of saline).     

-Transfer the 0.1 ml from each dilution tubes to the corresponding agar plates by spread plate technique.

-  Incubate the plates  at 37 ͦ c  for 24 hours

- following the incubation number of cells will be counted.

- Repeat this process from initial time of inoculation (0^th hour) to 7-8 times for every 30 min.  

- Calculate the CFU each time and the results will be tabulated and plotted the graph.

- To determine the generation time by using the formula Gt = t2 - t1  (CFU2 -CFU1).

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

CFU calculation:

No. of CFU / volume plated (ml) x total dilution used    to     No. of CFU/mL

Nutrient Agar Composition:

Peptone   5g

Beef extract   2g

Yeast extract    3g

Sodium chloride   5g

Agar   15 g

Distilled water   1000ml

Saline  Preparation: 

0.9 gm of Nacl in 100ml of water.

Measurement of Microbial growth by turbidometric method

Objective:

To find the generation time of given organism by turbidity method.

Principle:

Turbidometric method is the rapid, sensitive and useful technique to measure the growth of microbes. Turbidometry is based on the fact that microbial cells scatter light striking them. Since the microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells and indirectly related to cell number. One visible characteristic of growing bacterial culture is the increase in turbidity (cloudiness) of the medium.  When the concentration of bacteria increasing, the medium appears slightly cloudy or turbid. Further increase in concentration results in greater turbidity. When a beam of light is passed through a turbid culture, the amount of light transmitted is measured, Greater the turbidity, lesser would be the transmission of light through medium. Thus, light will be transmitted in inverse proportion to the number of bacteria. Turbidity can be measured using spectrophotometer.

Materials required:

-Nutrient broth

- 10- 12 hrs old broth culture

-Spectrophotometer

-Cuvette

Procedure:

-  Prepare and take 50ml of nutrient broth in sterile flask.

- Then this broth is inoculated with 1 ml of overnight bacterial culture and places it in incubator.

- The 0^th hour reading take by using spectrophotometer and the culture is again keep it in incubator.

- The flask is removing from incubator for a particular interval time (30 min.) and the sample is transfer aseptically to the cuvette using sterile pipette for observe the reading.

- The optical density reading in spectrophotometer is at 620nm.

- The reading will be tabulated and plotted the graph by OD value in Y-axis and incubation time in X -axis.  

Determination of generation time:

-Select the 2 points on the optical density scale that represents doubling the turbidity.

 The generation time is to be determine by using the formula

 Gt = t (OD2) –t(OD1)

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

Peptone Broth Composition:

Peptone    20g

Sodium chloride    5g

Distilled water   1000ml

Direct Microscopic count

Objective:

To determine the concentration of bacterial cell in a given sample by direct microscopic count method and calculating the generation time of the cell.

Principle:

Direct microscopic counts are performed by spreading a measured volume of sample over a known area of a slide, counting representative microscopic fields, and relating the averages back to the appropriate volume-area factors. Specially constructed counting chambers, such as the Petroff-Hauser chambers and Heamocytometer, simplify the direct counting procedure because they are made with depressions in which a known volume overlies an area that is ruled into squares. The ability to count a defined area and convert the numbers observed directly to volume makes the direct enumeration procedure relatively easy. Direct counting procedures are rapid but have the disadvantage that they do not discriminate between living and dead cells.

The ruled area of the hemocytometer consists of several large 1 x 1 mm (1mm² ) squares, which are subdivided in three ways; 0.25 x 0.25 mm (0.0625 mm²), 0.25 x 0.20 mm (0.05 mm²) and 0.20 x 0.20 mm (0.04 mm²). The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05 mm (0.0025 mm²) squares. Hold the cover slip(  0.1 mm)  at the raised edges of hemocytometer, which gives each square a defined volume.

 

	Area	Volume at 0.1mm depth
1 x 1 mm	1 mm2	100 nl
0.25 x 0.25 mm (1/16)	0.0625 mm2	6.25 nl
0.25 x 0.20 mm (1/20)	0.05 mm2	5 nl
0.20 x 0.20 mm (1/25)	0.04 mm2	4 nl
0.05 x 0.05 mm (1/400)	0.0025 mm2	0.25 nl

                                            

Materials required:

-Uniform bacterial cell suspension in peptone/nutrient broth

-Haemocytometer

-Coverslip

-Micropipette

-Microscope

 Procedure:

-Preparation uniform cell suspension:  Take cell suspensions in test tube and pipette out up and down for several times to get accurate cell counting and uniform distribution of the cell.

-Place a coverslip over the calibrated surface of the counting chamber.

- Using a pipette, transfer the suspension to the groove of the counting chamber to fill the chamber by capillary action.

-To observe the cells by using high magnification in the microscope, and count the number of cells in at least 5 of the small squares.

-Do the bacterial cell counting by using above methods, from 0^th hour upto several times for every 30 minutes to find out the generation time of cells.

-Take cell suspension for every time from culture flask which is incubated at 37 ͦ C

-Use the following formula to calculate the number of cells and plot the graph to find out the generation time of particular microbial cells

               Bacteria/mm³ = (bacteria/square) (25 squares) (50) (10³) 

Result:

The generation time of ­­­_______cells found to ­­­­­________ min/hr.

Peptone Broth Composition:

Peptone    20g

Sodium chloride    5g

Distilled water   1000ml

Nutrient Broth Composition:

Peptone   5g

Beef extract   2g

Yeast extract    3g

Sodium chloride   5g

Distilled water   1000ml