Methods Of Isolation And Concentration Estimation Of Microorganisms

What is the specific purpose of the study? 

What materials were used? 
How were they used? 
Where and when was the work done? (This question is most important in field studies.) 

Spread Plate Technique

According to Sagar Anyal (2016), “Spread plate technique is the method of isolation and enumeration of microorganisms in a fixed culture, and distributing it evenly.” The technique separates microbes contained within a small sample volume which is spread over the surface of an agar plate, using a sterile L-shaped glass rod to ensure even distribution of the colonies in order for them to form evenly on the agar surface. Small beads may also be used in place of the L-shaped glass rod to evenly spread the sample over the agar plate to allow for uniform growth of the culture. The plate that is used in spread plating is required when dry and when it is at room temperature, in order to ensure that the agar is able to absorb the microbes especially bacteria more rapidly. The spread plate technique is used for viable plate counts, moreover, it is also used to calculate the cell concentration in the test tube, from which the sample was plated, and finally it is used in the enrichment, selection and screening of experiments. Enrichment is important in the case where the microbe under study exists with another microbe that tends to overgrow I the culture and hence to suppress the growth of the unwanted microbe, the desired microbe is enriched with nutrients that enable it to grow in large colonies. Despite the numerous uses, the method faces some limitations which include: the method favors the rapid growth of aerobes and facilitates slow growth of micro aerophilic organisms. Also, the colonies in this method cloud and therefore this makes enumeration quite difficult.

Pour plate method on the other hand is a method that involves the counting of the number of colonies forming the microorganisms that are present in the liquid specimen. In this method, the microbes grow both on the surface and within the medium. According to Tankeshwar (2016), the colonies that grow within the medium are generally small sized colonies that may be confluent whereas the few microbes that grow in the surface of the medium (agar), are of the same size and appearance as those on the streak plate. The number of microorganisms present in this particular test sample is determined by the formula:

Colony Forming Unit (CFU)/ml = CFU × dilution factor × 1/ aliquot

The pair plate method is more efficient in counting the bacteria, however, it gives lower counts because heat sensitive microbes may die when they come into contact with the molten agar. 

Pour Plate Method

The difference between the pour plate technique and the surface spread method are that for the pour plate method, the microorganisms are in a liquid sample whereas for the surface spread method, the microorganisms are in solid samples. In addition to that, for pour plate method, the cultures grow within the media and on the surface on the media whereas for the surface spread method, the culture grows only on the surface of the media. This indicates that the pour plate favors the growth of both anaerobic and aerobic microbes whereas the surface smear method only favors the growth of aerobic microbes that require air for growth.  

https://microbeonline.com/pour-plate-method-principle-procedure-uses-dis-advantages/

The most probable technique on the other hand estimates the concentration of viable microorganisms in a sample through the replication of liquid broth growth in ten-fold dilution (Nisha Rijal, 2017). The method estimates the microbial populations in water, soils and agricultural products. This technique has numerous advantages, some of which are the ease of interpretation of the results, dilution of toxins present in the sample solution and the method`s suitability for analyzing turbid sample like mud, which cannot be analyzed by membrane filtration. However, the method has various limitations such as: it is time consuming, some of the results may not be accurate, there might be possibilities of false positives in the technique and lastly, the method requires a lot of hardware such as glass and also requires a lot of media.

According to Lumen Learning, direct counting methods like the use of haemocytometer enables the determination of bacterial concentration without the use of advanced equipment. The methods involve counting of cells in a known culture volume in order to obtain the concentration of the microbes. The haemocytometer works by creating a volumetric grid divided into differently sized cubes for counting the number of microorganisms in the cube accurately and thereafter, obtaining the sample`s concentration. Dilution of the cultures is required before the plating. Spectrophotometry is another method that can be used to calculate the cell concentrations by measuring the changes in turbidity. The spectrophotometer can also be used to measure the transmittance of solutions and also measure the diffusivity of any range of light. In spectrophotometry, the cultures do not need to be diluted but they should be above a given cell density. The turbidimetric methods measure the cell mass indirectly because the cell cultures are turbid as they absorb some light and allow the rest of the light to pass through. Higher cell concentrations result in higher turbidity. This report focuses on the methods of isolation of the microbes as well as acquiring the concentrations of the cultures. These methods are applicable in identifying specific microorganisms in plants, treatment of water sources and diagnosing diseases in other organisms.

Pair Plate Method

Materials and Methods

1. Preparation of dilute series (Day 1)

Materials per pair

• Overnight culture of coli
• 9 sterile test tubes
• 100ml bottle of quarter strength peptone water solution as the diluent.
• Sterile 1-5ml tips
• Sterile 0.1-1ml tips
• 3 nutrient agar plates
• 3 nutrient agar deeps held molten at 48⁰C
• 15 tubes of nutrient broth
• 3 sterile plastic spreaders
• 1-5ml Finn pipette
• 1-1ml Finn pipette

9.0 ml of peptone water solution was put in each of the nine sterile test tubes using sterile 5ml tip and the test tubes were thereafter labelled from 10^-1 to 10^-9. 1ml of E.coli suspension was added to the test tube labelled 10^-1 using a sterile 1ml tip which was later discarded. The contents in the test tube 10^-1 were thereafter mixed by vortexing for ten seconds. 1.0ml of this 10^-1 dilution was thereafter transferred to the second tube, thus, effecting a 10^-2 dilution in the second tube. The other dilution series were then prepared in a similar manner.

1. b) Surface Spread Method

Materials.

• Inoculated media from day 1
• Colony counters
• NA plate
• 1ml sterile tip
• Sterile spreader

0.1ml of 10^-7 dilution was transferred to the center of a well-dried NA plate using a fresh sterile 1ml tip. This liquid drop was then spread over the surface of the agar plate using a sterile spreader. 0.1ml of 10^-6 dilution and 0.1ml of the 10^-5 dilution were transferred using the same sterile tip to different plates and each were spread out in a similar manner. The colony counts were scored on all the plates and thereafter, calculation of the colony forming units per milliliter of the original suspension was done.

1. c) Pour Plate Method

Materials

• Inoculated media from day 1
• Colony counters
• Sterile petri dish
• 0ml sterile tip
• Nutrient agar

1ml of the 10^-8 dilution were transferred to a sterile petri dish using a fresh sterile 1.0 ml tip. This same pipette tip was used to transfer 1.0 ml of the 10^-7 and 1.0 ml of the 10^-6 dilutions to sterile petri dishes. The tip was then discarded and thereafter, nutrient agar was added to each of the three dishes and the mixture was thereafter mixed thoroughly by carefully and gently rotating the dish. The colony counts were then scored on all the plates and thereafter, calculation of the colony forming units per milliliter of the original suspension was done.

1. d) Most Probable Number Technique

Materials

• Inoculated media from day 1
• Colony counters
• Sterile bottles
• 1ml sterile tip

1ml of the 10^-9 dilution was transferred to each of the 5 bottles of the NB while using a fresh sterile 1ml tip. The same was done for the 10^-8 and 10^-7 dilutions. The number of bottles that were inoculated from each dilution and which showed turbidity were recorded. From the supplied most probable number technique table, the viable cell count of the original suspension was estimated.

Direct Microscopic/ Breed smear

Materials

• Slides and cover slips
• Broth culture of Saccharomyces cerevisiae
• 1cm²area measure
• Finnpipettes and 0.200 microlitres sterile yellow tips

An area of 1 cm² was marked on a glass slide. Thereafter, 10 microlitres of the Saccharomyces cerevisiae were then dried and fixed over a flame, stained with crystal violet for 30 seconds, rinsed and allowed to dry. While the smear dried, the microscope factor was prepared by setting a 10 × eyepiece and oil immersion objective and using the stage micrometer to measure the field diameter. Thereafter, the area of the field and the microscope factor were calculated. The smear was then examined by using the oil immersion lens and data was recorded. Afterwards, the microscope factor and average number of organisms were used to calculate the number of organisms in the original broth.

Most Probable Technique

Materials

• Helios Epsilon Spectrophotometer
• Broth culture of Saccharomyces cerevisiae
• Sterile Malt Extract Broth
• 5 test tubes and rack
• 1 test tube containing 4 ml of Malt Extract Broth for adjusting 100% T
• Sterile 1-5 ml white tips
• 1-5 ml Finn pipette

4 ml of the Saccharomyces cerevisiae culture was put in a test tube and was undiluted. Another 4 ml of the Saccharomyces cerevisiae culture was put in a test tube containing 4 ml of the sterile malt extract broth. The mixture was shaken to allow for the contents to mix thoroughly. Afterwards, 4 ml was taken from the newly formed mixture and added to another test tube that contained 4 ml of the sterile malt extract broth to form a dilution of the ratio 1:4. The process of dilution was repeated to make dilutions of ratios: 1:8 dilution and 1:16 dilution. Later on the readings for each test tube were taken using the Helios Epsilon Spectrophotometer. A graph of absorbance against the tube numbers was then prepared.

The power button at the back of the instrument was turned on and the instrument was allowed to perform a “power-up” sequence for two minutes. The MODE button was pressed until absorbance (A) was selected and displayed on the screen. The wavelength for the experiment was thereafter chosen and the tube containing 4 ml of sterile malt exchange agar was inserted into the sample holder and the blank was set to an absorbance of 0.00. The blank was then removed and the undiluted solution test tube, the 1:2, 1:4, 1:8 and 1:16 dilution test tubes were inserted sequentially into the sample holder and each of their absorbance was measured and recorded.

Materials

• Haemocytometer
• Coverslip
• Pasteur pipette

Procedure

A coverslip was placed over the central section. Thereafter, a drop of diluted cell suspension was placed on the sample introduction point. The cells were then allowed to settle for 3 minutes and were then counted using high dry (40 ×) objective lens. The number of organisms in the original broth were calculate using the haemocytometer depth and the area of the counted cells. The results were then recorded.

RESULTS: VIABLE COUNTS METHOD:

1) SURFACE SPREAD:

DILUTION	NUMBER OF COLONIES
10-6	>300
10-7	152
10-8	120

Surface spread method can be calculated by using the following formula:

Number of cells × inverse dilution × 10 cfu/ml

Here, 120 colonies are yielded by plate of 10^-8 dilution. So the calculation is:

120× 10^-8× 10 = 0.000012 cfu/ml 1.2 × 10^-5 cfu/ml 

POUR PLATE:

DILUTION	NUMBER OF COLONIES
10-7	102
10-8	47
10-9	77

 Pour plate method can be calculated by using the following formula:

Number of cells × inverse dilution × 1 cfu/ml

Plate of 10^-7 dilution yields a count of 102 colonies. So the calculation is:

 47× 10^-8×1= 0.00000047

Haemocytometer Method

MOST PROBABLE NUMBER:

DILUTION	NUMBER OF TUBES SHOWING GROWTH
10-7	5
10-8	5
10-9	4

The probability table shows that 5, 5 and 4 number of tubes showing growth with 10^-7, 10^-8 and 10^-9 dilutions yields 1600 numbers of viable cells. So the calculation is:

1600× 10⁶ = 169600 cells/ml

RESULTS: TOTAL COUNTS:

1. TURBIDIMETRIC METHODS:

Absorbance	Dilution
0.777A	Undiluted
0.512A	1:2
0.224A	1:4
0.102A	1:8
0.004A	1:16

Fig.1) The graph representing dilution versus absorbance in the sample.

The curve shows that more the sample will be diluted, lesser would be its absorbance (optical density).

1. BREED SMEAR:

1).Calculate the average number of organisms per field of view.

The cells observed in one field was= 89 cells per field

The cells observed in second field was=72 cells per field

Total no. of cells= 161

Average no. of organisms can be calculated as: =89+72=   161=   80.5 cells/ mL

                                                                                 2             2

2). Calculate the microscope factor:

The formula of the area of the field of view is given by: A =    p r²

                                                                                     : A =  p (0.08)² =0.02 mm²

                                   Microscope factor = 100

                                                       Area of field (mm)

                                                             = 100 / 5,000

                                                               = 0.02

3). Calculate the number of organisms in the original broth:

Organisms in original broth can be calculated by using the microscope factor and the average number of organisms per field of view such as:

      No. of cells = 80.5

Microscope factor = 5000

No. of organisms into original broth= 80.5 × 5000 =4.02 ×10⁵

Field of view considered =2, dilution sample =1:2

 No. of cells in original broth=4.02 ×10⁵ ×100 ×2 =8.04× 10⁷ cells/ mL 

C). HAEMOCYTOMETER:

 Calculate the number of organisms in the original broth/mL.

Number of cells observed = 2

Volume of 1ml= 1000 mm³

Area under investigation= 0.004 sq. mm

So, number of cells ×1000 /0.004 = 2 × 1000 / 0.004

                                                        = 500,000 cells 

Discussion and Conclusion

In the experiment two major methods to count bacterial population in sample were used. Each of these methods produced different results. For viable counts method, Surface spread method yielded 120 colonies with (0.000012 cfu/ml) by multiplication with 10^-8 dilution and volume  and shown better results than other dilutions 10^-6 as the colony was more than 300 and hence were to be disregarded. The pour plate technique of dilution of 10^-7 yields 102 colonies (0.102cfu/ml) (by multiplying with inverse of dilution and volume)which were countable but discrete colonies in other dilutions (Harrigan 2014) were less such as only 47 and 77 as a result of the clustering of cells that mainly occurs in this technique and hence, causes inaccuracy in viable cell counting. In addition, some microbes that may have been heat sensitive may have died in the procedure of this method, lowering the cell count. For the MPN, the examined 5 tubes with the dilutions each showed maximum growth and from the MPN table, the number of microbes that were present were 1600. For total count method, the turbidimetric method showed the curved graph by using spectrophotometer and taken the readings at 680 nm wavelength to measure the concentration of sample. It showed that an increase in number of cell concentration indicated an increase in the turbidity of solution. Thus, the absorbance increased with increase in the cell concentration as this theory was suggested by Beer Lambert (Nelson 2013). These instrumental methods are more proficient for counting the number of cells which are hardly visible. The Breed Smear is a method that measures the concentration of dead and live cells and the results showed that 8.04× 10⁷ cells/ mL were present in the solutions. For the Haemocytometer, the cells that were present on the right and left side lines were counted and results into 500,000 cells. The experiment was successful as the aims of the experiments were all reached.

References

Aryal, S. (2016) Spread Plate Technique-Principle Procedure and Uses. Microbiology Info. Online. Available from: https://microbiologyinfo.com/wp-content/uploads/2016/10/Spread-Plate-Technique-Principle-Procedure-and-Uses.pdf [Retrieved 10 May, 2018].

Tankeshwar (2016) Pour Plate Method: Principle, Procedure, Uses and (Dis) Advantages. Microbe Online, Online. Available from: https://microbeonline.com/pour-plate-method-principle-procedure-uses-dis-advantages/  [Retrieved 10 May, 2018].

Rijal, N. (2017) Most Probable Number (MPN) Test: Principle, Procedure and Results. Microbe Online. Online. Available from: https://microbeonline.com/probable-number-mpn-test-principle-procedure-results/  [Retrieved 10 May, 2018].

Lumen Learning Temperature and Microbial Growth. Boundless Microbiology. Online. Available from: https://courses.lumenlearning.com/boundless-microbiology/chapter/temperature-and-microbial-growth/ [Retrieved 10 May, 2018].

 Harrigan, W. F. a. (2014). Laboratory Methods in Microbiology, Saint Louis : Elsevier Science.

Nelson, D. L. (2013). Lehninger principles of biochemistry, New York : W.H. Freeman.