LAB5: Determination of Antimicrobial Effects of Microbial Extracts

Introduction

Pathogenic and spoilage microorganisms had created many undesired effects to human race. Antimicrobials are used to overcome these problems. Antimicrobial is a group of drugs includes antibiotics, antifungals, antiprotozoals, and antivirals that are used to destroy microorganisms or to prevent their development. They are used in various aspects such as in agriculture, surgery, treatment and food production. Some examples of antimicrobials which is critical important to human are Penicillins, Aminoclycosides and Streptogramins.

Lactobacillales or lactic acid bacteria (LAB) are microorganisms that produce lactic acid and proteinaceous bacteriocins which inhibit other microorganisms to survive. They are gram-positive, acid-tolerant, non-sporeforming cocci, coccobacilli or rods and lack catalase. All LAB grow anaerobically, but unlike most anaerobes, they can grow in the presence of O₂ as "aerotolerant anaerobes". LAB bacteriocins contribute to the taste and texture of fermented products and inhibit food spoilage bacteria by producing growth-inhibiting substances and large amounts of lactic acid as acidification inhibits the growth of spoilage agents. They are mainly used in food processing such as making yogurt, cheese, cultured butter, sour cream and sausage. Some examples of Lactobacillales are Lactobacillus, Lactococcus and Streptococcus.

Objective

To determine the antimicrobial effects of extracellular extracts of selected LAB strains

Materials and reagents

MRS broth

Sterile filter paper disk (50mm x 50mm)

Forceps

Sterile universal bottles

Cultures of LAB and spoilage/pathogenic organisms

Bench-top refrigerated centrifuge

Incubator

UV/Vis spectrophotometer

Distilled deionized water

Trypticase soy agar (TSAYE)

Brain heart infusion agar (BHI)

Yeast extract

Procedure

(Refer to lab manual)

Results

Part 1: Determination of bacteriocin activity via agar diffusion test

Strains of LAB	Strains of spoilage/pathogenic bacteria	Inhibition zone(cm)
LAB 1	Staphylococcus aureus	No
LAB 2	Staphylococcus aureus	No

Part 2: Determination of bacteriocin activity via optical density

Dilutions	OD600 of spoilage/pathogenic bacteria
	Strain : Staphylococcus aureus
	Reading 1	Reading 2	Reading 3	Average
0x	0.453	0.445	0.476	0.458
2x	0.671	0.665	0.673	0.670
10x	0.970	1.014	1.004	0.996
50x	1.090	0.995	0.950	1.012
100x	0.887	0.948	0.945	0.927
Equation				y = 0.0031x + 0.7115
OD600 of control	0.247	0.225	0.229	0.234
50% of OD600	0.124	0.113	0.115	0.117
AU/ml				-191.774

Discussion

Part 1: Determination of bacteriocin activity via agar diffusion test

From this experiment, the volume of the LAB extracellular extract and MRS needed for each universal bottle is shown as below:

Dilution Mixture	0x	2x	10x	50x	100x	Control
LAB Extracellular Extract (ml)	5.0	2.5	0.5	0.1	0.05	0
MRS Broth (ml)	0	2.5	4.5	4.9	4.95	5.0
Total (ml)	5.0	5.0	5.0	5.0	5.0	5.0

Antimirobials are the substance that produced by a certain microorganism to prevent other microorganisms to compete resources with them. In this experiment, bacteriocin, lacic acid, diacetyl and hydrogen peroxide act as the antimicrobials which is produced by Lactic Acid Bacteria (LAB) are tested by antimicrobial susceptibility testing to test their ability to inhibit the growth of SA (Staphylococcus aureus) culture. The goals of antimicrobial susceptibility testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections. The effectiveness of LAB antimicrobials towards the growth of SA can be determined by measuring the inhibition zones around the LAB-staining paper disks. Inhibition zones are the places where the growth of a certain microorganism is inhibited by antimicrobial. The larger the inhibition zones, the higher the degree of sensitivity of SA to LAB antimicrobial.

However, in our experiment, the inhibition zones around the LAB paper disks cannot be determined as there is no growing of SA culture. This is probably caused by the concentration of SA spoilage culture is too dilute. In our experiment, we mixed 2 ml of SA culture into 100 ml of BHI agar but refer to our lab manual, we should mix 20 ml of SA culture into 100 ml of BHI agar. The low concentration of SA culture causes their growth in agar is totally suppressed by LAB antimicrobials. This results we cannot measure the degree of sensitivity of SA to LAB antimicrobial. Thus, we can at least speculate there is a suppression of LAB to SA culture as SA cannot grow in the agar with LAB. Besides, the bacteriocins produced by the lactic acid bacteria(LAB) may not strong enough.

Part 2: Determination of bacteriocin activity via optical density

Optical density can be measured by using the spectrophotometer. Optical density is used as a measure of the concentration of bacteria in a suspension. The spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium; it is used to determine the amount of light scatter. When visible light passes through a cell suspension, the light is scattered. Greater degree of scatter proves that more bacteria are present.

A spectrophotometer can be set at a wavelength of 420-660nm. Normally the OD600 is measured. OD600 is an abbreviation indicating the optical density of a sample measured at a wavelength of 600 nm. OD600 is also preferable to UV spectroscopy when measuring the growth over time of a cell population. It is because at this wavelength, the cells will not be killed as they would under too much UV light.

spectrophotometer

One arbitrary (AU) is defined as the dilution factor of the extracellular extract that inhibited 50% of the   spoilage/pathogenic bacteria growth and expressed as AU/mL.

Abs600 = Z. Thus, 50% of Z = Z/2

y = mx + c; Thus, x = (y-c)/m

When y = Z/2, Thus x = (Z/2 - c)/m

From the experiment, the strain we used is Staphylococcus aureus. It is a gram-positive coccal bacterium that is frequently found in the respiratory tract and on the skin.

The result we obtained shows that the optical density decreases slightly when the dilution is 100 x. However, the graph still shows the positive gradient. This indicates the positive inhibition on the growth pattern of the bacteria. It means the higher the concentration of extracellular extract, the higher concentration of bacteriocin, the lower the growth rate of bacteria. The inaccurate result might due to human error, we did not use the pipette correctly that causes the volume of MRS and Staphylococcus aureus taken incorrect. Besides, when we transfer the extracellular extracts into the mixture, we should take the supernatant instead of pellet, if not it might affect the accuracy of result. The expectation of the result is when the serial dilution increasing, the optical density increasing too. The graph plotted should be in linear positive gradient.

Conclusion

LAB is used to produce bacteriocin which can inhibit the growth of bacteria (antimicrobial effect). Nowadays, the usage of LAB is getting attention as it exerts a strong antagonistic activity against many microorganisms, including food spoilage organisms and pathogens. The graph of determination of bacteria activity via optical density showing a positive gradient indicates positive inhibition of Staphylococcus aureus results in high optical density.

References

https://en.wikipedia.org/wiki/OD600

http://www.ifrj.upm.edu.my/20%20(01)%202013/17%20IFRJ%2020%20(01)%202013%20Noordiana%20(017).pdf

http://cid.oxfordjournals.org/content/49/11/1749.full

LAB4 : SOURCES OF CONTAMINATION AND INFECTION

Introduction

Contamination is the unwanted pollution of something by another substance, for example leaking of petroleum storage tanks in the sea. There are three main causes of contamination: physical, chemical and microbiological hazards. Many harmful microorganisms can be transmitted by a host and in air currents with dust particles. When a victim or a host was infected by a diesease, he or she can easily spread the infectious microorganism into air by coughing, sneezing or even breathing. Thus, it is very difficult to be avoided to come into contact with these microorganisms.

Every human contacts with two types of microorganisms, which is resident and transient microorganisms. Resident microorganisms are life-long members of the body's normal microbial community, but are not found everywhere. They are normally colonised on the surface of the skin, mucous membranes, digestive tract, upper respiratory system and distal portion of the urogenital system throughout life. They are usually commensal that feeds on excreted cellular wastes and dead cells or mutualistic by provide vitamin K and Vitamin B.

Transient microorganisms are Normal Microbiota remain in the body for only hours to months before disappearing. They cannot persist in the body because pf the competition from resident microorganisms, elimination by the body's defense cells, and chemical or physical changes in the body that expel them. These microorganisms contact with human in daily life such as breathing, eating and touching. Resident and transient microbes cause the host no harm to human under normal circumstance. They become opportunistic pathogens only

when immune suppression of the host, reduction in microbial antagonism, and introduction of normal microbiota into an unusual site in the body.

Objective

To determine the microorganisms in the air and from healthy humans

Materials and Reagents

Molten nutrient agar

Sterile water

Sterile petri dishes

Sterile clinical swab

Pipette and tips

Bunsen burner

Procedure

(Refer to lab manual)

Results

1. Air

	Commercial molten agar	Self-made molten agar
Elevation	raised	raised
Margin	entire	Entire,undulate
Texture	moist	moist
Size	Small,large	small
Form	circular	Circular,irregular
Surface	Smooth,rough	smooth
Colour	Pale yellow	white

2. Normal breathing

	Commercial molten agar	Self-made molten agar
Elevation	Flat,raised	raised
Margin	entire	entire
Texture	moist	moist
Size	small	small
Form	circular	circular
Surface	Shiny and smooth	smooth
Colour	yellow	white

3. violent couging

	Commercial molten agar	Self-made molten agar
Elevation	Raised,convex	raised
Margin	Entire, undulate	entire
Texture	dry	dry
Size	small	small
Form	Circular,irregular	circular
Surface	Shiny, smooth	Shiny, smooth
Colour	yellow	yellow

4. Ear

	Commercial molten agar	Self-made molten agar
Elevation	raised	raised
Margin	entire	entire
Texture	moist	moist
Size	small	small
Form	Circular,irregular	Circular,irregular
Surface	Shiny and smooth	Shiny and smooth
Colour	white	white

5. Hand

	Commercial molten agar	Self-made molten agar
Elevation	raised	raised
Margin	Entire,undulate	Entire,undulate
Texture	moist	moist
Size	Small,large	Small,large
Form	Circular,irregular, filamentous	Circular,rhizoid, irregular
Surface	Shiny&smooth, rough&dull	Smooth, rough&dull
Colour	Yellow,white	white

Discussion

Bacterial populations grow extremely fast when under the favourable environmental condition and adequate nutrients condition. Different types of bacteria will produce colonies that have different appearances. Colony morphology is a way scientists identify bacteria according to their size, surface, texture, colour, form, elevation and margin.

For the contamination by air, there were little or fewest colonies on the both media compared to the other media that we have prepared. It is due to the low concentration of contaminants in the air of laboratory. The common bacteria found in the air are Bacillus, Staphylococci and Clostridium.

For contamination by hand, there were many colonies formed. This is because our hand carries thousands of microbes as we came in contact with many contaminated things throughout our living.

For ear contaminations, the sample was taken by an ear pick. Before it came in contact with our ear, the ear pick was dipped into distilled water .The sample is taken from the outer part of our ear and is spread on the culture by using streaking plate technique. As we could see from the result, the colonies were formed along the line that we streaked. Some common bacteria that lives in our outer part of the ear are Staphylococcus,Turicillotitidis and Alloiococcusotitis. For the internal part of the ear, most of the microbes were trapped by ceremonious gland.

For violent coughing, we could observe only few colonies on both agars. This is because most of the bacteria and microbes from nasal passage being trapped by the mucus and expelled to the outer environment as we cough.

For normal breathing, there are a few spots of colonies formed. Most of the bacteria from are non-pathogenic but some of them may cause illness. From normal breathing of a healthy person, the bacteria that might present are Staphyloccocus, Epedermis ,Corynebacterius and Micrococcus Luteus.

Conclusion

From the experiment, we found out that there are not significant differences between the colonies formed in commercial agar and self-made agar. Bacteria can grow well as long as the conditions are favourable. From the results observed, we also can concluded that the organs exposed to the outer environment, like ears and hands will contain more bacteria. At the end, we able to identify the bacteria according to the characteristics of colony morphology.

Reference

https://wikis.engrade.com/a105science2012/agarplating

http://labreport102.blogspot.my/2014/12/lab-report-4.html

http://ibg102.blogspot.my/2014/12/lab-4-sources-of-contamination-and.html