Sunday 20 May 2012

LAB 5 SHAHRUL EZZATI SHAHRUL AMIR -111416-

INTRODUCTION
Multiwavelength ultraviolet/visible (Uv-vis) spectroscopy is a versatile,quantitative, rapid, and reliable analytical tool that has immediate applications as a biosensor for the detection, identification, and enumeration of microorganisms and cells. The sample information contained in a spectrum includes cell size, chemical composition, shape, and information on their internal structure.  This information is obtained from the spectroscopic analysis of a sample measured over a broad range of wavelengths (200-900 nm) and/or with the scattered light measured at one or many different angles. The potential to extract  large amounts of information from a single multiwavelength measurement makes Uv-vis spectroscopy a powerful characterization tool. In addition, process Uv-vis spectrometers and miniaturized systems, make this technique readily available for real-time in situ monitoring of biological, and environmental processes.
Bacteriocins comprise a large and diverse group of ribosomally synthesised antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have receive special attention in recent years due to their poential application in the food industry as natural biopreservatives. Different classes of LAB bacteriocin have been identified on the basis of biochemical and genetic characterization.

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

RESULTS

PART 1 : Determination of bacteriocin activity via agar diffusion test

STRAINS LAB
Strains of spoilage / pathogenic bacteria
Inhibitio zone ( cm )

L.plantanum
S.aureus
0.0
K.pneumonia
0.9
P.aeruginose
0.9

L.casei
S.aureus
0.0
K.pneumonia
0.7
P.aeruginose
0.7

L.brevis
S.aureus
0.0
K.pneumonia
0.6
P.aeruginose
0.7


LAB : L.plantanum
K.pneumonia


P.aeruginosa

S.aureus
LAB : L.casei
K.pneumonia
P.aeruginosa
S.aureus



LAB : L.brevis
K.pneumonia


P.aeruginosa

S.aureus
PART II :Determination of bacteriocin activity via optical density


Dilutions
OD600 of spoilage/pathogenic bacteria
S.aureus
K.pneumonia
P.aeruginosa
0X
-
-
-
2X
0.454
0.484
0.410
10X
0.610
0.696
0.620
50X
0.386
0.571
0.312
100X
0.238
0.464
0.265
Equation
y = 0.013x+0.278
y = 0.113x+0.111
y = -0.031x+0.532
OD600  of control
0.270
0.892
0.432
50% of OD600
0.135
0.446
0.216
AU/ml
-11
2.96
10.19



DISCUSSION
Multiwavelength Uv-vis spectra of microorganisms and cell suspensions contain quantitative information on their properties such as number, size, shape, chemical composition, and internal structure. These properties are essential for the identification and classification of cells.  The complexity of microorganisms in terms of their chemical composition and internal structure make the interpretation of their spectral signature a difficult task. In this lab report  proposed for the interpretation of the multiwavelength spectra of microorganisms.
The optical properties as functions of wavelength, and available literature data on the size and chemical composition of E. coli cells and S. aureus have been used to explore the sensitivity of the calculated spectra to the model parameters. It is shown that the proposed model can reproduce the features of experimentally measured spectra. The sensitivity of the spectra to the model parameters suggests that the proposed model can be used for the quantitative deconvolution of the Uv-vis spectra in terms of critical information necessary for the detection and identification of microorganisms.
An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body.
A drug used to treat a microbial infection. "Antimicrobial" is a general term that refers to a group of drugs that includes antibiotics, antifungals, antiprotozoals, and antivirals.

CONCLUSION
A model for the interpretation of the multiwavelength Uv-vis spectra of microorganisms and cells has been formulated. The proposed interpretation model is based on light scattering theory, spectral deconvolution techniques, and on the approximation of the frequency dependent optical properties of the basic constituents of living organisms.

LAB 4 : SHAHRUL EZZATI BT SHAHRUL AMIR 111416


INTRODUCTION
A microorganism or microbe is an organism that is unicellular or lives in a colony of cellular organisms. Microorganisms contamination is a situation which occurs when microorganisms end up in a location where they are not supposed to be. It is often used to refer to contamination of food by bacteria which can cause disease, but microorganisms contamination can also occur in other settings. This situation is not desirable, because it can pose a health threat and cause other problems. As microorganisms, particularly bacteria, are found practically everywhere, this means in most cases the reduction of harmful microorganisms to acceptable levels. However, in some cases it is required that an object or substance be completely sterile, i.e. devoid of all living entities and viruses.
Although a number of microorganisms are present in air, it doesn't have an indigenous flora. Air is not a natural environment for microorganisms as it doesn't contain enough moisture and nutrients to support their growth and reproduction.
In food preparation microorganisms are reduced by preservation methods (such as the addition of vinegar), clean utensils used in preparation, short storage periods or by cool temperatures. If complete sterility is needed, the two most common methods are irradiation and the use of an autoclave, which resembles a pressure cooker. There are no conditions where all microorganisms would grow, and therefore often several different methods are needed.

OBJECTIVE
To determine the microorganisms in the air and from healthy human

RESULT
NORMAL BREATHING

VIOLENT COUGHING

AIR
HAND
EAR

DISCUSSION
Air currents may bring the microorganisms from plant or animal surfaces into air. These organisms may be either commensals or plant or animal pathogens. However, the transmission of animal diseases is not usually important in outside air.
The main source of airborne microorganisms is human beings. Their surface flora may be shed at times and may be disseminated into the air. Similarly, the commensal as well as pathogenic flora of the upper respiratory tract and the mouth are constantly discharged into the air by activities like coughing, sneezing, talking and laughing. The microorganisms are discharged out in three different forms which are grouped on the basis of their relative size and moisture content. They are droplets, droplet nuclei and infectious dust.
Everyone knows that flu viruses and cold germs can congregate on hands. Also found on the hands are the bacteria that cause gastrointestinal problems. One of the most common types of bacteria known to man is staph. Because in recent years staph is becoming increasingly resistant to antibiotics which can prove deadly, washing your hands is more than good hygiene, it can save your life.
The respiratory system is open to airborne microbes and to outside pollution.It is not surprising that respiratory diseases occur, in spite of the body's defenses. Some respiratory disorders are relatively mild and, unfortunately, very familiar. We all experience the excess mucus, coughing, and sneezing ofthe common cold from time to time. The common cold is an example of rhinitis, an inflammation of the epithelium lining the nose and nasal cavity. Viruses, bacteria, and allergens are among the causes of rhinitis. Other respiratory disorders include laryngitis, pneumonia, bronchitis, chronic obstructive pulmonary disease, and lung cancer.
The cough reflex is a vital part of the body's defence mechanisms. Normally, the lungs and the lower respiratory passages are sterile. If dust or dirt get into the lungs, they could become a breeding ground for bacteria and cause pneumonia or infection in the breathing tubes. When a person coughs, there is a short intake of breath and the larynx (the voice box) closes momentarily. The resulting blast of air comes out at high speed, scrubbing and clearing the airway of dust, dirt or excessive secretions.
CONCLUSION
Bacteria are everywhere and can spread from surface to surface, person to person, food to food, and person to food. Harmful bacteria can be controlled by practicing the 4 Cs of food safety. To prevent the spread of harmful bacteria, proper cleaning of both hands and surfaces is especially important.  The good thing is that not all bacteria are harmful; most bacteria are beneficial to us.
When designing experiments, you should always use safe techniques when working with bacteria. Also, it's important to have a control plate. In this experiment, you also learned that different strains of bacteria can be identified through colony morphology.

Saturday 5 May 2012

LAB 5 MOHAMMAD SYAFIQ BIN ABDULLAH 113569


LAB 5: DETERMINATION OF ANTIMICROBIAL EFFECT OF MICROBIAL EXTRACT

INTRODUCTION
     An antimicrobial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body.
     The history of antimicrobials begins with the observations of Pasteur and Joubert, who discovered that one type of bacteria could prevent the growth of another. They did not know at that time that the reason one bacterium failed to grow was that the other bacterium was producing an antibiotic. Technically, antibiotics are only those substances that are produced by one microorganism that kill, or prevent the growth, of another microorganism. Of course, in today's common usage, the term antibiotic is used to refer to almost any drug that attempts to rid your body of a bacterial infection. Antimicrobials include not just antibiotics, but synthetically formed compounds as well.
     The discovery of antimicrobials like penicillin and tetracycline paved the way for better health for millions around the world. Before penicillin became a viable medical treatment in the early 1940s, no true cure for gonorrhea, strep throat, or pneumonia existed. Patients with infected wounds often had to have a wounded limb removed, or face death from infection. Now, most of these infections can be cured easily with a short course of antimicrobials.
     However, with the development of antimicrobials, microorganisms have adapted and become resistant to previous antimicrobial agents. The old antimicrobial technology was based either on poisons or heavy metals, which may not have killed the microbe completely, allowing the microbe to survive, change, and become resistant to the poisons and/or heavy metals.
     Antimicrobial nanotechnology is a recent addition to the fight against disease causing organisms, replacing heavy metals and toxins and may some day be a viable alternative.
Infections that are acquired during a hospital visit are called "hospital acquired infections" or nosocomial infections. Similarly, when the infectious disease is picked up in the non-hospital setting it is considered "community acquired".
     Certain groups of bacteria can produce antimicrobial substances with the capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Interest in naturally produce antimicrobial agents, such as bacteriocins, is on the rise, since nowadys consumers demand "natural" and "minimally processed" food.
     Bacteriocins comprise a large and diverse group of ribosomally synthesized antimicrobial proteins or peptides. Although bacteriocins can be found in numerous Gram-positive and Gram-negative bacteria, those produced by lactic acid bacteria (LAB) have received special attention in recent years due to their potential application in the food industry as natural biopreservatives. Different classes of LAB bacteriocins have been identified on the basis of biochemical and genetic characterization. These bacteriocins have been reported to inhibit the growth of Listeria monocyotogenes,Staphylococcus aureus, Enterococcus faecalis and Clostridium tyrobutyricum.

OBJECTIVE

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

RESULT

PART 1 : Determination of bacteriocin activity via agar diffusion test

figure 1
 presence of antimicrobial effects

figure 2
 presence of antimicrobial effects

STRAINS LAB
Strains of spoilage / pathogenic bacteria
Inhibitio zone ( cm )

L.plantanum
S.aureus
0.0
K.pneumonia
0.9
P.aeruginose
0.9

L.casei
S.aureus
0.0
K.pneumonia
0.7
P.aeruginose
0.7

L.brevis
S.aureus
0.0
K.pneumonia
0.6
P.aeruginose
0.7


PART 2 : Determination of bacteriocin activity via optical density

Dilutions
OD600 of spoilage/pathogenic bacteria
S.aureus
K.pneumonia
P.aeruginosa
0X
-
-
-
2X
0.454
0.484
0.410
10X
0.610
0.696
0.620
50X
0.386
0.571
0.312
100X
0.238
0.464
0.265
Equation
y = 0.013x+0.278
y = 0.113x+0.111
y = -0.031x+0.532
OD600  of control
0.270
0.892
0.432
50% of OD600
0.135
0.446
0.216
AU/ml
-11
2.96
10.19




DISCUSSION
     The lactic acid bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics. These bacteria, usually found in decomposing plants and lactic products, produce lactic acid as the major metabolic end-product of carbohydrate fermentation. This trait has, throughout history, linked LAB with food fermentations, as acidification inhibits the growth of spoilage agents. Proteinaceous bacteriocins are produced by several LAB strains and provide an additional hurdle for spoilage and pathogenic microorganisms.
     Furthermore, lactic acid and other metabolic products contribute to the organoleptic and textural profile of a food item. The industrial importance of the LAB is further evinced by their generally recognized as safe (GRAS) status, due to their ubiquitous appearance in food and their contribution to the healthy microflora of human mucosal surfaces. The genera that comprise the LAB are at its core Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weisella; these belong to the order Lactobacillales.

PART 1


The agar diffusion test, or the Kirby-Bauer disk-diffusion method, is a means of measuring the effect of an antimicrobial agent against bacteria grown in culture.
The bacteria in question is swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases.
     If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.
     Kirby-Bauer antibiotic testing (KB testing or disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria are grown on agar plates in the presence of thin wafers containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition).
     This along with the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity to that particular antibiotic. In general, larger zones correlate with smaller minimum inhibitory concentration (MIC) of antibiotic for that bacteria. This information can be used to choose appropriate antibiotics to combat a particular infection.

PART 2

     Optical density that is measured in the spectrophotometer can be used as the measurement of the concentration of the bacteria suspension. It is basically used for the measurement of transmittance or reflectance of solution that are depending on their wavelength.
        Optical density of this experiment contain MRS medium and pathogenic bacteria was determined by using the spectrophotometer of OD600.  One from the others type of LAB used is Lactobacillus plantirum. While the tree type of pathogenic microorganism that had been used were S.Aureus, P.Aeruginosa and K.Pneumonia.
        The positive-controls that show higher optical density than the other sample show antimicrobial effect. While the negative-control was prepared in MRS with 2mL distilled water is for auto-zero via spectrophotometer.

CONCLUSION
     Bacteriocins from lactic acid bacteria are of importance in bioconservation of various foods. Moreover, the use of more than one LAB bacteriocin as a combination of biopreservative may have major applications in improving food safety. In the present study, the inhibitory effect of the cell-free filtrates of each of the 20 isolates was evaluated. Antimicrobial activity was observed for 6 isolates, and only against Gram positive bacteria. The biochemical nature of the antibacterial molecule produced by S. thermophilus T2 was studied in both the cell-free supernatant and the chloroform extract.

REFERENCE
http://en.wikipedia.org/wiki/Lactic_acid_bacteria