Friday, 19 December 2014

Lab 5:  Determination of Antimicrobial Effects of Microbial Exracts

INTRODUCTION
There are a few groups of bacteria that can produce antimicrobial substances. An antimicrobial is an agent that kills microorganism or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibacterials are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Organic acids, hydrogen peroxide, diacetyl and bacteriocins are included among these antimicrobial compounds. Since nowadays consumers demand “natural” and minimally processed” food therefore there is a rising of interest on naturally produced antimicrobial agents like bacteriocins.
      Bacteriocins are proteins or complexed proteins biologically active with antimicrobial action against other bacteria, principally closely related species. They are produced by bacteria and are normally not termed antibiotics in order to avoid confusion and concern with therapeutic antibiotics, which can potentially illicit allergic reactions in humans and other medical problems
      Bacteriocin production could be considered as an advantage for food and feed producers since, in sufficient amounts, these peptides can kill or inhibit pathogenic bacteria that compete for the same ecological niche or nutrient pool. This role is supported by the fact that many bacteriocins have a narrow host range, and is likely to be most effective against related bacteria with nutritive demands for the same scarce resources
      Lactic acid bacteria (LAB) are characterized as Gram-positive cocci or rods, non-aerobic but aerotolerant, able to ferment carbohydrates for energy and lactic acid production. LAB bacteriocins can work via different mechanisms to exert an antimicrobial effect, but the cell envelope is generally the target. The initial electrostatic attraction between the target cell membrane and the bacteriocin peptide is thought to be the driving force for subsequent events. 

Materials and Reagents

MRS broth
Sterile filter paper disk
Forceps
Sterile universal bottles
Cultures of LAB and spoilage/pathogenic organisms
Bench-top refrigerated centrifuge
Incubator 30oc and 37oc
UV/V is spectrophotometer
Distilled deionized water
Trypticase soy agar
Brain heart infusion agar
Yeast extract

PROCEDURE

Part 1: Determination Of Bacteriocin Activity Via Agar Diffusion Test

1. All the petri dishes are labelled according to the spoilage organisms and strains on LAB used.
2. Each plate was only used for one strain of spoilage organism and one strain of LAB. Plate was divided into 2 parts, each for on replicate.
3. Each group has 1 strain of LAB and 1 strain of spoilage/pathogenic organism.
4.10ml of trypticase soy-yeast extract agar (TSAYE) was loaded into the labeled petri ish and the agar was ensured to fully cover the entire surface of the plate. It is waited until it solidifies.
5. 2ml of broth containing the spoilage organism was innoculated into 10ml of brain hear infusion (BHI) agar and vortex.
6. The mixture was loaded on top of the TSAYE agar layer and ensurd that it covered the entire surface and waited to solidify.
7. The broth containing the lab cultures was centrifuge. The supernatent was used as a extracellular extracts.
8. A sterile filter paper disk is picked up aseptically with a sterile forcep and a disk is dipped into the extracellular extract.
9. The paper disk was placed on top of the solidifies BHI agar.
10. The plates were inoculated for 24-28 hours at 37oC.
11. Upon incubation, the inhibition zones were measured (in cm) and is recorded.

Part 2: Determination Of Bacteriocin Activity Via Optical Density

1. The broth was containing LAB cultures were centrifuge. The supernatent is used as extracellular extracts.
2. Each group has 1 strain of LAB and 1 strain of spoilage/pathogenic organism.
3. 5 ml of double-strength MRS was added with 1 ml of cultures containing spoilage/ pathogenic bacteria and the mixture was vortex.
4. A serial dilution of the extracellular extracts (diluted 0x, 2x, 10x, 50x, 100x) were prepared.
5. 5 ml of each extracellular extracts dilution was added into mixture as prepared in step(3).
6. The mixtures were incubated for 12-15 hours at 37ºC.
7. A control using 5 ml of double-strength MRS, 1 ml of cultures containing spoilage/ pathogenic bacteria, and 5 ml of sterile peptone was prepared. The mixtures were incubated for 12-15 hours at 37ºC.
8. A negative control for ‘auto zero’ via the spectrophotometer was prepared. 5 ml of double-strength MRS was added with 2 ml of distilled water. (Need not to be incubated)
9. Upon incubation, the optical density of the spoilage/ pathogenic bacteria at 600 nm was measured. The same was performed for the control as well.
10. One arbitrary unit (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.
11. 50% of the spoilage/ pathogenic bacteria growth were determined from the OD600 of the control.
RESULTS

Part 1: Determination Of Bacteriocin Activity Via Agar Diffusion Test
Presence of inhibition zone


Absence of inhibition zone

Strain of lab
Strain of spoilage/pathogenic bacteria
Name
Inhibition  Zone (cm)
Average
Lactobacillus fermentum
S.Aureus
sample 1
1 and 1.1
1.05
Lactobacillus fermentum
S.Aureus
sample 2
1 and 0.9
0.9
Lactobacillus fermentum
S.Aureus
sample 3
0.6 and 0.7
0.65
Lactobacillus fermentum
S.Aureus
sample 4
0.7 and 0.7
0.7
Lactobacillus fermentum
S.Aureus
sample 5
No inhibition zone
Lactobacillus fermentum
S.Aureus
sample 6
No inhibition zone




















Part 2: Determination of Bacteriocin Activity Via Optical Density

Dilution
OD600 of Spoilage/ Pathogenic Bacteria
0x
0.269
2x
0.397
10x
0.448
50x
0.174
100x
0.123
Equation
y= -0.0026x + 0.3678
OD600 of Contol
0.107
50% of OD600
0.0535
AU/ml
100/11= 9.091


 

DISCUSSION

Part 1: Determination Of Bacteriocin Activity Via Agar Diffusion Test
1. Bacteriocins are bactericidal, antibiotic-like substances, apparently protein in nature, which are produced by many bacteria and have a killing action on strains of the same or closely related species.
2.The larger the inhibition zone(no bacteria growing area) on the agar medium,means that the bacteriocin is effective on the pathogenic bacteria and vise versa.
3. Type of the LAB of bacteriocin  that being used is Lactobacillus fermentum and the bacteria that we used is S.Aureus.
4.The bacteriocin will cause the destruction of the membrane potential by forming the pores on the pathogenic bacteria. It will cause the destruction of the membrane potential by forming the pores on the pathogenic bacteria.It will inhibits the nucleolytic activity of the pathogenic bacteria strains which breaks down the DNA chromosomes as well as RNA.Then the bacteriocin will inhibits the protein synthesis of the pathogenic bacterias but does not kills them.
5.As for the none inhibition zone exist result are because not enough Lactobacillus fermentum are being aplied  around the pathogenic bacteria.This is because without adequate numbers of Lactobacillus fermentum, the point of critical mass which is needed cannot occur and the bacteria will be unable to have the desired impact on the symptoms being treated.

Part 2: Determination Of Bacteriocin Activity Via Optical Density
1.Optical density, measured in a spectrophotometer, can be used as a measure of the concentration of bacteria in a suspension. As visible light passes through a cell suspension the light is scattered. Greater scatter indicates that more bacteria or other material is present. The amount of light scatter can be measured in a spectrophotometer. Typically, when working with a particular type of cell, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will want to use cells that are in their mid-log phase of growth. Typically the OD600 is measured.
2. 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.
Control : 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
3. As the serial of dilution increse, the optical density will increase to indicate that the Lab which is Lactobacillus Plantarum has strong microbial effec on the pathogenic bacteria which is E.coli.
4. The control value of the experiment is 0.209 and the value of the absorbant is 0.102.
5. From the result,the graph shows that as the serial dilution increases, the optical density decreases. This shows that there is negative inhibition of the pathogenic bacteria. This might be caused by the using of distilled water during the process of serial dilution. As we know, the distilled water is colourless. When the Lactobacillus Plantarum is diluted with distilled water, the optical colour density will become very much lower compared to the normal colour density of Lactobacillus Plantarum culture. So, the results obtained is wrong. 
6. Therefore, the peptone is suggested to replace with the distilled water in the serial dilution of   Lactobacillus Plantarum culture as the colour of peptone is quite similar with the culture. 

CONCLUSION
LAB is a useful bacteria used to produce bacteriocin that can inhibit the growth of bacteria.The use of strains that produce multiple bacteriocins could be advantageous to limit the potential emergence of bacteriocin-resistant populations An important aspect to take into consideration in relation to the commercial use of bacteriocins is the tolerance or resistance of certain pathogenic bacterial species that are normally sensitive since it may compromise the antibacterial efficiency of these compounds

REFERENCES

http://www.academia.edu/3745035/Bacteriocins_Nature_Function_and_Structure

http://www.scielo.br/scielo.php?pid=s1516-89132007000300018&script=sci_arttext

http://nootriment.com/lactobacillus-fermentum/


http://people.hofstra.edu/beverly_clendening/adv_molecular_biology/Protocols/Measuring_Optical_Dens.html

Monday, 8 December 2014

Lab 4 : Sources of Contamination and Infection

Introduction 

Airborne particles are a major cause of respiratory ailments of humans, causing allergies, asthma, and pathogenic infections of the respiratory tract. Airborne fungal spores are also important agents of plant disease, and the means for dissemination of many common saprotrophic (saprophytic) fungi.
Here we consider some important respiratory diseases of humans,the roles of airborne spores in crop diseases and the methods used to monitor spore populations in the air.
Air sampling is used routinely to monitor the populations of airborne particles, and to inform the public about air quality and pollen/spore counts through public broadcasting. It is used  to monitor the populations of specific allergenic particles. And it is used in crop pathology for disease-forecasting, so that growers can apply fungicides as and when required.


 Material
Molten nutrient agar
Sterile water
Sterile petri dishes
Sterile clinical swab
Pipette and tips

Procedure


Air:
1.Molten agar was poured into the sterile dish and cooled.
2.The lid was removed from the plate and the lid was leave for resting on the side of the plate.
3.The lid was replaced and incubated at 37C for 48 hours.

Hands:
1. Hand was washed using sterile water. Soap was used.
2. 1ml of wash water was transfered to petri dish using an automatic pipette.
3. Molten nutrient agar was added to the petri dish.
4. The lids of the Petri dish was replaced and the dish was gently rotated until the wash water is thoroughly mixed with the molten agar. Agar did not com in contact with the lid of the dish.
5. After the agar has set, the dish is inverted and incubated at 37oC for 48 hours.

Ear:
4. Molten agar was poured into sterile petri dish and cool.
5. Using extreme care, a sterile swab moistened with sterile isotonic solution is rubbed into the ear of the subject.
6. The swab is used to inoculate the labeled plate.
7. Incubated at 37oC for 48 hours.

Normal breathing:
1. Molten agar is poured into sterile petri dish and cooled
2. The lid was removed and the plate was held about 15 cm from your mouth. Breathe normally but directly onto the plate for one minute. Lid is replaced back onto the petri dish.
3. Incubated at 37oC for 48 hours.

Violent coughing:
1. Molten agar was poured into sterile petri dish and cool.
2. Lid was removed and plate was held about 15 cm from your mouth. Cough violently onto the agar. Lid was replaced .
3. Incubated 37oC for 48 hours.
Result

Discussion 
After the result obtained,we can see various types of calony marphology.there are few basic characteristics of colony marphology that are typically evaluated.  These are the characteristics of colony marphology:
As we use different types of media which are prepared natrium broth and commercial broth, the colony morphology will differ for both types of the media. 

Morphology of Bacteria Colonies of Ear 

                     Commercial molten agar              Prepared molten agar      
Elevation:   Raised                                             Raised
Form       :   Circular                                          Circular, Irregular      
Surface  :    Shiny and Smooth                          Shiny and Smooth
Texture  :    Dry                                                  Dry      
Colour    :    White, Buff, Yellow                       Buff, Yellow
Margin    :   Entire                                             Entire,


Morphology Bacteria Colonies of Normal Breathing
 
                    Commercial molten agar              Prepared molten agar      
Elevation:   Raised, flat, crateriform                   Raised
Form       :   Circular, Irregular,                           Circular
Surface  :   Shiny and Smooth                           Shiny and Smooth
Texture  :   Dry                                                   Dry      
Colour    :   Buff, Yellow                                     Yellow     
Margin    :   Entire, undulate                              Entire

Morphology of Bacteria Colonies of Violent Coughing
 
                   Commercial molten agar              Prepared molten agar    
Elevation:  Raised,convex                                   Raised
Form       :   Circular                                           Circular,filamentous
Surface  :   Shiny and Rough                            Shiny and Smooth
Texture  :   Dry                                                   Dry                
Colour    :   Yellow                                            yellow, buff         
Margin    :   Entire                                               Entire, Lobate

Morphology of Bacteria Colonies of Air

 Prepared Molten Agar:
Elavation : Raised
Form : Circular,irregular,filamentous
Surface: shiny,smooth
Texture : dry
Colour : Yellow, buff
Margin : Entire, undulate, filiform


Morphology of Bacteria Colonies of Hand
 
                   Commercial molten agar               Prepared molten agar
Elevation: Raised,flat                                         Raised, crateriform
Form       :  Circular, Irregular                            Circular, Rhizoid, Irregular                
Surface  :  Shiny and Smooth                              Shiny and Smooth, Dull and Rough     
Texture  :  Dry                                                     Dry                
Colour    :  Buff,yellow                                      Yellow, buff
Margin    :  Entire, Lobate                                  Entire, undulate

Leading research indicates there are many more individual species of bacteria on the hands than scientists once thought. Most of these bacteria are harmless, but they can cause disease if they enter the body through a break in the skin. The main pathogenic bacteria which are more likely to be found on the hands are Staphylococcus, Corynebacteria, Streptococcus, Myobacteria, and Haemophilus.

Because the ear is exposed to the outside environment, despite the best efforts of the ceruminous glands, the healthy outer ear still houses a variety of microbes. Some of the most common bacteria are Staphylococcus epidermis, Turicellaotitidis, Alloiococousotitis, Pseudomonas aeruginosa, Corynebacterium, Staphylococcus aureus, and Streptococcus saprophyticum. The most common fungal microbe known to reside in the ear is Candida albican

Our mouth contains pathogenic and non-pathogenic microorganisms. Some examples of these non-pathogenic bacteria are Streptococcus, Neisseria, Haemophilus, and Micrococcus. The pathogenic bacteria are Corynebacterium diphtheriaeStreptococcus pyogenes,Staphylococcus aureus and Streptococcus pneumoniae. Whereas the pathogenic bacteria might cause strep throat, scarlet fever, diptheria

Air also contains microorganisms. There are vegetative cells and spores of bacteria, fungi and algae, viruses and protozoan cysts. Air mainly is transport or dispersal medium for microorganisms.  The most common genera of fungi in indoor air are Penicillium Aspergillus. For bacteria, the common genera found in indoor air are Staphylococci, Bacillus and Clostridium.

Our mouth is a home to bacteria, viruses, fungi, and protozoa. The species of Staphylococcus most often found in the mouth include Staphylococcus epidermidis and Staphylococcus aureus. Streptococcus mutans, Streptococcus mitis, Streptococcus salivarius, Streptococcus pneumoniae and Streptococcus pyogenes all live in the mouth.  Lactobacillus bacteria also present in mouth. While most of the rod-shaped E. coli in the body is located in the intestines, a small amount of the bacteria is present in the mouth. Actually the microbes during breathing and coughing are quite similar because there are originated from the same place, mouth and nose cavity in human beings.
 

Conclusion
1. There is no significant difference between the commercial agar and own prepared nutrient agar except for a difference in the type of bacteria's in the colony

2.The observations show that our human outer skin surface grows more bacteria than the inner body. The exhale air has about the same contamination as the atmospheric air.

2. We learned that there different types morphology types in bacteria


References 
  • https://microbewiki.kenyon.edu/index.php/Ear
  • http://www.livestrong.com/article/201160-types-of-bacteria-on-childrens-hands/
  • http://en.wikipedia.org/wiki/Contamination
  • http://www.urmc.rochester.edu/encyclopedia/content.aspx?ContentTypeID=1&ContentID=2089