ISOLATION AND ANTIMICROBIAL SCREENING OF BACTERIA FROM SOIL OBTAINED FROM TWO DIFFERENT LOCATION IN SULTAN IDRIS EDUCATION UNIVERSITY, PERAK.

SBL1023

TECHNIQUE IN BIOLOGY AND BIOCHEMISTRY LABORATORY

TITLE:

ISOLATION AND ANTIMICROBIAL SCREENING OF BACTERIA FROM SOIL OBTAINED FROM TWO DIFFERENT LOCATION IN SULTAN IDRIS EDUCATION UNIVERSITY, PERAK.

LECTURER’S NAME:

PROFESSOR MADYA DR. ROSMILLAH BINTI MISNAN

GROUP MEMBER	MATRIC NO.
THAM WING YEW, NICHOLAS	E20172019166
NURUL SYAWAL BINTI MOHD YAZI	E20172019183
NUR SYAHIRAH BINTI AB HALIM	E20172019173
NUR ADILLAH BINTI MOHAMMAD	E20172019191
NURUL HIDAYAH BINTI SALIM	E20172019197

TITLE

Isolation and antimicrobial screening of bacteria from soil obtained from two different locations in Sultan Idris Education University, Perak.

ABSTRACT

Actinobacteria, sometimes known as soil bacteria, is one of the contributing sources of antibiotics till the present days. In this research, total of 20 strains were grown on nutrient agar from the dilution of samples (samples are collected from surface of the soil and 10 cm depth) obtained from two different locations in Sultan Idris Education University, Perak. From these 20 plates, only 4 plates were chosen for the calculation of colony forming unit (cfu) based on the number of colonies formed on the plates. Plates that have too little or too many colonies are rejected for calculation of cfu and are not suitable to be used for the research. From these 4 plates, spreading of the colonies were done on other nutrient agar and 14 plates of isolates were obtained at last. Then, gram staining test is done and only gram-positive bacteria are used for the evaluation of inhibitory activity against 5 strains of microorganism (Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Saccharomyces cerevisiae and Staphylococcus aureus). One finding of this research is the bacteria from the agar plug resist the insertion of the pathogens, but do not secrete chemicals to create inhibition zone, which means that the growth of the pathogen is higher, dominating the nutrient agar (NA) or the bacteria from the agar plug also grow together but in different colonies of a small quantity on the same nutrient agar. 

INTRODUCTION

Generally, bacteria are present at our surrounding, on our phone, table, door knob and the list goes on. It is a misconception, stereotypical and old-fashioned thought that all bacteria are harmful. Indeed, some bacteria are essential in terms of their medicinal purposes in such way that it helps to fight infections by producing antibiotics. These antibiotic-producing bacteria is utmost important in the medical field especially in combatting resistant pathogens.

An antimicrobial is an agent that kills microorganisms or stops their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibiotics are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Agents that kill microbes are called microbicidal, while those that merely inhibit their growth are called biostatic.

In this study, bacteria from soil obtained from two different locations in Sultan Idris Education University, Perak will be isolated and tested for their bioactive compounds and selected isolates will be identified using standard method.

A huge number of currently used antibiotics including erythromycin, streptomycin, rifamycin and gentamycin, are all products isolated from soil actinomycetes. The richness and diversity of actinomycetes present in any specific soil, is greatly influenced by the soil type, geographical location, cultivation and organic matter amongst other factors. Numerous studies have been done by scientists to isolate actinomycetes, as sources of antibiotics. However, as actinomycetes occur widely in nature, only a small percentage of the globe and a small proportion of actinomycetes species have been screened. Therefore, the present study is aimed to isolating antibiotic producing actinomycetes strains from other soil.

OBJECTIVES

1. To isolate bacteria from soil obtained from two different locations in Sultan Idris Education University.

2. To screen antimicrobial activities of bacteria isolated.

MATERIALS

• Soil sample

• Nutrient broth

• Nutrient agar

• Distilled water

• Glycerol

• Ethanol

• Crystal violet

• Iodine

• Safranin

• Immersion oil

• Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Saccharomyces cerevisiae and Staphylococcus aureus strains

APPARATUS

• Centrifuge tubes

• Spatula

• Weighing bottle

• Measuring cylinder

• Analytical balance

• Micropipette

• Micropipette tips

• Pipette and pipette filler

• L-shaped spreader

• Universal bottle

• Sterile petri dish

• Beaker

• Hot plate with magnetic stirrer

• Bunsen burner

• Slide

• Inoculating loop

• Disposable gloves

• Microscope

• Forceps

• Screw-capped cryotube

• Media bottle

METHODOLOGY

1. Soil Sample Collection

First, soil sample are collected from two different locations in Sultan Idris Education University, Perak. The coordinates for the sampling locations are 3.7210799, 101.5243979 and 3.7216482, 101.5251493. Soil of depths 0 cm and 10 cm are obtained from these locations.

2. Isolation of bacteria

10^-1/2 suspensions of environmental samples are collected at Sultan Idris Education University, Perak which is 2g of soil from different depths of 0 cm and 10 cm. The samples are prepared using nutrient broth. The resultant suspensions are sonicated, shaken and 10^-1 to 10^-5 dilutions are prepared in nutrient broth. Aliquots of each dilution (0.1ml) is spread duplicate, onto the isolation media listed in Table 1. All of the plates are left to dry for 30 minutes in a laminar flow hood prior to incubation, as recommended by Vickers and Williams (1987).  In addition, soil particles which is 2g, are sprinkled directly over the isolation media. The inoculated plates are incubated at 28°C and 37°C for up to one month. The various actinobacterial colony types growing on the selective isolation plates are observed and recorded. Each bacteria colony is subcultured onto nutrient agar plates, using sterile toothpicks, and incubated at 28^oC for 14 days prior to preparing stock cultures.

3. Colony Forming Unit of Bacteria

Then, the total number of bacteria growing on the isolation plates are calculated and expressed as the mean number of cfu’s per gram dry weight environmental sample using the formula:

cfu/ml = (number of colonies) x dilution factor

amount plated (ml)

4. Morphological observation and gram staining

Bacteria isolates are subcultured onto nutrient agar and incubated at 28^oC for 14 days. The morphology of the bacteria grown on the nutrient agar is observed. Coccus bacteria are spherical or oval in shape, like a berry and the bacillus bacteria are rod-like in shape. The isolates from the plates are tested for gram staining to determine whether the colonies are of gram-positive bacteria or gram-negative bacteria.

5. Preparation of stock culture

From isolation experiment, bacteria colony types are taken from the selective isolation plates and subcultured onto yeast extract-malt extract agar (nutriet agar), using sterile toothpicks, and incubated at 28^oC for two weeks. Sterile glycerol is prepared and transferred into sterile cryotube. Biomass of the isolates grown on nutrient agar for two weeks at 28^oC are used to prepare duplicate glycerol (20% w/v) stocks in screw-capped cryotubes. One of the glycerol stocks, the working culture, is stored at -20^oC; the remaining two are kept at -80°C for long term preservation.

6. Antimicrobial activity of bacteria isolates

Isolates are grown on both nutrient agar for 14 days at 28˚C. Antimicrobial screening is achieved using a standard agar plug assay (Fiedler, 2004) against wild type strains of Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Saccharomyces cerevisiae and Staphylococcus aureus. Agar plugs are taken from the cultures using the back of sterile blue tips and arranged in Petri dishes. Bacterial and yeast lawns are prepared by inoculating 100µL of overnight cultures grown in Luria Bertani broth into a medium containing 100mL of Luria Bertani broth and nutrient agar, mixing well and pouring into the square Petri dishes that contain the plugs prepared from the nutrient agar plates. The plates are incubated at 37˚C overnight and examined for the presence of inhibition zones around the agar plugs. The diameters of the inhibition zones are recorded.

RESULT

20 isolation plates.

        

Figure 1.  Isolation of bacteria

           

Table 1. List of the 20 plates of isolation of bacteria

CODE	LOCATION	DEPTH	DILUTION (ml)
U1	1	0	10-1
U1	1	0	10-2
U1	1	0	10-3
U1	1	0	10-4
U1	1	0	10-5
U1	1	10	10-1
U1	1	10	10-2
U1	1	10	10-3
U1	1	10	10-4
U1	1	10	10-5
U2	2	0	10-1
U2	2	0	10-2
U2	2	0	10-3
\ U2	2	0	10-4
U2	2	0	10-5
U2	2	10	10-1
U2	2	10	10-2
U2	2	10	10-3
U2	2	10	10-4
U2	2	10	10-5

From the 20 plates, only 4 plates are calculated for their CFU. This is because only these plates have good and suitable number of colony of bacteria for the calculation and do not have too much of bacteria. All of these plates have the number of colonies of bacteria below 60. The formula that is used to calculate the CFU is as following:

cfu/ml = (number of colonies) x dilution factor

amount plated (ml)

Table 2. The list of plates for CFU calculation which are only 4 plates are calculated,while  

              others are too numerous to count. (TNTC)

CODE	LOCATION	DEPTH	DILUTION (ml)	CFU/ml                        TNTC                (Too numerous to count)
U1	1	0	10-1	TNTC
U1	1	0	10-2	TNTC
U1	1	0	10-3	TNTC
U1	1	0	10-4	3.0x10-5
U1	1	0	10-5	TNTC
U1	1	10	10-1	TNTC
U1	1	10	10-2	TNTC
U1	1	10	10-3	2.9x10-4
U1	1	10	10-4	TNTC
U1	1	10	10-5	TNTC
U2	2	0	10-1	TNTC
U2	2	0	10-2	TNTC
U2	2	0	10-3	TNTC
\ U2	2	0	10-4	5.5x10-5
U2	2	0	10-5	TNTC
U2	2	10	10-1	TNTC
U2	2	10	10-2	TNTC
U2	2	10	10-3	TNTC
U2	2	10	10-4	2.0x10-5
U2	2	10	10-5	TNTC

CALCULATION:

1. U1, Location 1 (0cm) 11/1/19: 10^-4

CFU = 30 x 10^-4

          1

          =3.0 x 10^-5

2. U1, Location 1 (10cm) 11/1/19: 10^-3

CFU = 29 x 10^-3

          1

          =2.9 x 10^-4

3. U2, Location 2 (0cm) 11/1/19: 10^-4

CFU = 55 x 10^-4

           1     

             =5.5 x 10^-5

4. U2, Location 2 (10cm) 11/1/19: 10^-4

CFU = 20 x 10^-4

          1

          =2.0 x 10^-5

The plates are chosen to do the spreading method using Nutrient Agar (NA) after a week. Some of the plates are duplicated or triplicated for the best result.

At last, 14 plates are obtained from the spreading method and gram staining test is performed for each of it.

Table 3. The list of plates for spreading method using Nutrient Agar (NA). Some of the plates

              were duplicate, triplicate to 2 or 3 sample.

CODE	LOCATION	DEPTH	DILUTION (ml)
U1 a	1	0	10-4
U1 b	1	0	10-5
U1 c	1	0	10-5
U1 d	1	0	10-5
U1 e	1	10	10-3
U1 f	1	10	10-3
U1 g	1	10	10-4
U1 h	1	10	10-4
U1 i	1	10	10-5
U2 j	2	0	10-2
U2 k	2	10	10-3
U2 l	2	10	10-4
U2 m	2	10	10-4
U2 n	2	10	10-5

From the gram staining test, it is observed that the bacteria are of either bascillus or coccus based on the microscope.  

From the gram staining method, some of the bacteria were bascillus and coccus shape after being observed from microscope.

  

                      

Figure 2 Gram staining method

Table 4. The list of plates for gram staining

CODE	LOCATION	DEPTH	DILUTION (ml)	GRAM  POSITIVE / NEGATIVE
U1 a	1	0	10-4	Gram Negative
U1 b	1	0	10-5	Gram Positive
U1 c	1	0	10-3	Gram Negative
U1 d	1	0	10-5	Gram Positive
U1 e	1	10	10-3	Gram Negative
U1 f	1	10	10-3	Gram Negative
U1 g	1	10	10-4	Gram Positive
U1 h	1	10	10-4	Gram Positive
U1 i	1	10	10-5	Gram Positive
U2 j	2	0	10-2	Gram Positive
U2 k	2	10	10-3	Gram Negative
U2 l	2	10	10-4	Gram Positive
U2 m	2	10	10-4	Gram Positive
U2 n	2	10	10-5	Gram Positive

So, these 5 plates are removed for the following step which is subculture after a week. The code for the subculture is the prefix K.

Table 5. The list of 9 plates that were subculture with new coding with morphology

					MORPHOLOGY
CODE	AGAR	DEPTH	DATE	DILUTION (ml)	CELL	COLONY
K1	NA	0	23/1/19	10-5	Bacillus	Pale yellow,rod-shaped
K2	NA	0	23/1/19	10-5	Coccus	Pale yellow,spherical
K3	NA	10	23/1/19	10-4	Coccus	Pale yellow,spherical
K4	NA	10	23/1/19	10-4	Bacillus	Pale yellow,rod-shaped
K5	NA	10	23/1/19	10-5	Coccus	Pale yellow,spherical
K6	NA	0	23/1/19	10-2	Bacillus	Pale yellow,rod-shaped
K7	NA	10	23/1/19	10-4	Bacillus	Pale yellow,rod-shaped
K8	NA	10	23/1/19	10-4	Bacillus	Pale yellow,rod-shaped
K9	NA	10	23/1/19	10-5	Coccus	Pale yellow,spherical

Figure 3. Nine plates that were subcultured

These data were observed its own colour and recorded according to National Bureau of Standards (NBS) Colour Name Charts (Kelly, 1958).

The colour of all this data were pale yellow.

Figure 4. The colour were  recorded according to National Bureau of Standards (NBS) Colour Name Charts (Kelly, 1958).

Antimicrobial screening were achieved from this data using standard agar plug assay (Fiedler, 2004) against wild type strains:

Table 5. The list of pathogen that were used

NO	STRAIN
1	Bacillus subtilis
2	Escherichia coli
3	Pseudomonas fluorescens
4	Saccharomyces cerevisiae
5	Staphylococcus aureus.

             

            

Figure 5. Antimicrobial screening using standard agar plug assay (Fiedler, 2004) against wild  

               type strains.

From observation, none of the agar plug showed positive result for the secretion of inhibition zone. The bacteria from the agar plug resist the insertion of the pathogens, but do not secrete chemicals to create inhibition zone, which means that the growth of the pathogen is higher, dominating the nutrient agar (NA) or the bacteria from the agar plug also grow together but in different colonies of a small quantity on the same nutrient agar.

DISCUSSION

From the result obtained for CFU, only 4 plates had their CFU determined due to the suitability on the number of colonies formed on the plates. These plates have colony number less than 60 while the other plates have either too little or too many colonies. These plates are rejected for the calculation of CFU and are not suitable to be used for the research.

Subsequently, out of the 14 isolate plates, only 9 plates which are tested for gram positive in gram staining. The plates which are tested for gram negative in gram staining are rejected. One justification that can be made is that gram positive bacteria have a greater volume of peptidoglycan (a polymer of amino acids and sugars that create the cell wall of all bacteria in their cell membranes), which is what makes the thick outer covering. This thick outer covering, or membrane, is capable of absorbing a lot of foreign material and from genetic modification, it can help for the storage of medicinal compounds to combat pathogens. From these 9 plates, the morphology of the bacteria that is observed is that 5 isolate plates consist of bacillus type bacteria meanwhile the other 4 consist of cocci type bacteria.

Another finding of this research is that none of the agar plugs obtained from the 9 plates have the ability to resist the 5 strains of pathogens introduced to it (Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Saccharomyces cerevisiae and Staphylococcus aureus). the bacteria from the agar plug resist the insertion of the pathogens, but do not secrete chemicals to create inhibition zone, which means that the growth of the pathogen is higher, dominating the nutrient agar (NA) or the bacteria from the agar plug also grow together but in different colonies of a small quantity on the same nutrient agar.

CONCLUSION

All the objectives of this project is fulfilled. First, all the group members know the procedure or more precisely, the correct work flow for the isolation of bacteria from soil obtained from two different locations in Sultan Idris Education University. Also, all the group members are able to screen antimicrobial activities of bacteria isolated despite the negative result obtained. Due to time constraint, the finding of our project is limited but there is room for improvement.

ACKNOWLEGMENT

We are truly grateful to our lecturer, Professor Madya Dr.Rosmilah binti Misnan for her guidance which helped us a lot in finishing our project. We are also thankful to our supervisor, Dr. Hamidah Binti Idris for the guidance and support which made this project possible for our group to work on and also for the encouragement she gave to us during this project. It is a big pleasure for us to be able to work under her supervision.

We are also sincerely thankful to Mr.Ali who assisted us until the end this project. We are really grateful because we managed to complete this project within the time given by our supervisor. Lastly, this project could not be completed without the effort and cooperation from our group members.

REFERENCES

Dale, J. W. & Park, S. F. (2004). Molecular Genetics OF BACTERIA (4th ed.). Chichester, West Sussex: John Wiley & Sons Ltd.

Falkinham, J. O., Wall, T. E., Tanner, J. R., Tawaha, K., Alali, F. Q., Li, C. & Oberlies, N. H. (2009). Proliferation of antibiotic-producing bacteria and concomitant antibiotic production as the basis for the antibiotic activity of Jordan’s Red Soils. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 75(9), 2735-2741. doi: 10.1128/AEM.00104-09

Fiedler, H. (2004). Screening for bioactivity In Bull A (Ed.), Microbial Diversity and Bioprospecting (pp. 324-335). Washington, DC: ASM Press. doi: 10.1128/9781555817770.ch30

Maloy, S. R., Cronan Jr., J. E. & Freifelder, D. (1994). Microbial Genetics (2nd ed.).  Sudbury, MA: Jones and Bartlett Publishers.

Sethi, S., Kumar, R. & Gupta, S. (2013). Antibiotic production by microbes isolated from soil. INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES AND RESEARCH. 4(8), 2967-2973. doi: 10.13040/IJPSR.0975-8232.4(8).2967-73

Sultan Idris Education University. (2018). Isolation and antimicrobial screening of actinomycetes from BRIS soil. Tanjung Malim, Perak: Dr. Hamidah bt Idris.

Tenover, F. C. (2006). Mechanisms of antimicrobial resistance in bacteria. THE AMERICAN JOURNAL of MEDICINE. 119(6A), S3-S10. doi: 10.1016/j.amjmed.2006.03.011

Vickers, J. C. & Williams, S. T. (1987). An assessment of plate inoculation procedures for the enumeration and isolation of streptomycetes. Microbios Lett., 36, 113-117.