Monday, May 08, 2006

antimicrobial activities of aloe vera gel and leaf













Contents:-

1) Introduction
2) Introduction to Aloe barbadensis
3) Constituents of Aloe vera
4) Properties of Aloe barbadensis constituents
5) Antimicrobial effect of Aloe barbadensis
6) Botanical differences among three major species of aloe
7) Recent research on antimicrobial action of aloe vera extracts

8) Preparation of aloe vera gel extract
9) Preparation of aloe vera leaf extract

10) Pseudomonas aeruginosa
11) Pathogenesis of Pseudomonas aeruginosa



12) Isolation of Pseudomonas aeruginosa
13) Identification of Pseudomonas aeruginosa

14) Spectrophotometrical analysis of antimicrobial susceptibility testing of aloe vera gel against Pseudomonas aeruginosa
1. Requirements
2. Procedure
3. Result

15) Coliforms
16) Isolation of Coliforms
17) Identification of coliforms

18) Spectrophotometrical analysis of antimicrobial susceptibility testing of aloe vera gel against coliforms.
1. Requirements
2. Rrocedure
3. Result

19) Pathogenesis of Staphylococcus aureus
20) Isolation of Staphylococcus aureus
21) Identification of Staphylococcus aureus

22) Spectrophotometrical analysis of antimicrobial susceptibility testing of aloe vera gel against Staphylococcus aureus
1. Requirements
2. Procedure
3. Result
23) How aloe vera can protect fruits, vegetables ( preventing foods from coliforms based spoilage)

24) Antimicrobial susceptibility testing of Pseudomonas aeruginosa against aloe vera leaf extract.

25) Antimicrobial susceptibility testing of Pseudomonas aeruginosa against aloe vera gel.

26) Antimicrobial susceptibility testing of Aspergillus fumigatus against aloe vera gel.

27) Antimicrobial susceptibility testing of against Aspergillus fumigatus aloe vera leaf extract.


28) Keratinophilic fungi
29) Evaluation of the antimicrobial activity of the aloe vera extracts on keratinophilic fungi
a. Materials
b. Procedure
c. Result
30) Microscopic evaluation of quantitative effect of aloe vera gel extract by hanging drop technique
a. Pseudomonas aeruginosa
b. Coliforms
c. Staphylococcus aureus
31) Determination of phenol coefficient of aloe vera gel ( anthraquinones)





INTRODUCTION:-




Disease, infection and other health related problems have always been “matter of undesire” to humans. Trials and use of chemical therapeutics aid is now the intense toll to get them cured. These causes various constipations and added side effects. But these chemical therapeutics is not only the way, as the ancient world i.e. Various ancient civilizations knew, the use of Mother Nature, as a divine curing and healing power. Our traditional knowledge reveals the treasure of nature for the benefit of mankind and their help. Various herbs and medicinal plants are found on earth. This is the power filled into them, trough which these are continuing to serve mankind. In modern scenario these potent plants and herbs are being harvested by the use of biotechnology. This takes the “responsibility” of making much effective useful, fast, economical use of resources among the best of “available or developed ways”.

One of such a small effort has been made by us to reveal the magnificent nature of aloe vera ( Aloe barbadensis), one among the known medicinal plant for its antimicrobial effect on some harmful micro-organisms and some other additional applications using the basic and simpler techniques of biotechnology.

In chattisgarh, the climate permits the facilitated growth of aloe vera. Some years ago these herbs were found in every home garden. The last decade resulted its depletion from its natural habitat. This plant being industrially useful and economical, resulted in very fast depletion from its native habitat of chattisgarh. This plant is now grown in the farms and the fields. The growth rate of this plant i.e. the natural propagation is very slow, therefore there exists an intense need to develop a technique to improve the growth rate of the plant. Also there exists many screened facts about the plant which are still to be revealed.


Aloe barbadensis (Aloe vera)
A succulent perennial plant belonging to the lily family, aloe vera grows wild in Madagascar and large portions of the African continent. Because of its many therapeutic uses, it is now commercially cultivated in the United States, Japan, and countries in the Caribbean and Mediterranean. Many individuals also grow aloe as a houseplant.
The aloe plant is best known for its healing aloe vera gel, a thin, clear, jellylike substance that can be squeezed or scraped from the inner part of the fleshy leaf. A soothing juice is also made from this gel.
Another substance from the same plant, aloe vera latex, is taken from specialized cells along the inner leaf skin (called the pericyclic tubules). The latex is extracted as a liquid, then dried into a yellow powder. Because it's such a potent laxative, the latex is not usually used alone but combined with gentler herbs, such as cascara sagrada. Germany's Commission E approves of using small amounts of aloe vera latex to relieve constipation, but only for short-term use.
Aloe Vera (Aloe barbadensis, A. capensis, A. vulgari) contains anthraquinones, polysaccharides (including glycoproteins and mucopolysaccharides) fatty acids, enzymes, prostaglandins, salicylates, minerals, vitamins, sterols, and amino acids.
The action of the latex portion, which contains the anthraquinones, is cathartic, bitter digestive stimulant, anthelmintic, digestive tonic (in small doses), purgative (in higher doses), and topically is a stimulant (irritant) and desiccant.
The action of the inner gel is Antibacterial, Antifungal, Antiviral, Immunostimulant, wound healer, Hypoglycemic, and Anti-inflammatory
Constituents of aloe vera :-
1. Vitamins
It is rich in all vitamins excluding Vitamin D, especially the antioxidant Vitamins A (beta-carotene), C and E and even contains a trace of Vit. B12, one of the very few plant sources of this vitamin. This is important for vegetarians and vegans.
2. Enzymes
Several different types of these biochemical catalysts when taken orally aid digestion by breaking down fat and sugars.
One in particular, Bradykinase, helps to reduce excessive inflammation when applied to the skin topically and therefore reduces pain, whereas others help digest any dead tissues in wounds. Lipases and proteases which break down foods and aid digestion are present.
3. Minerals
Calcium, Sodium Potassium, Manganese, Magnesium, Copper, Zinc, Chromium and the anti-oxidant Selenium.
Although minerals and trace elements are only needed in very small quantities, they are essential for the proper functioning of various enzyme systems in different metabolic pathways.
4. Sugars
These are derived from the mucilage layer of the plant which surrounds the inner gel. and are known as mucopolysaccharides, which enhance the immune system and help to detoxify. Aloe Vera contains both mono and polysaccharides, but the most important are the long chain sugars involving glucose and mannose or the gluco-mannans which I have already referred to. These sugars are ingested whole from the gut, not broken down like other sugars, and appear in the bloodstream in exactly the same form. This process is known as pinocytosis. Once in the blood stream they are able to exert their immuno-regulating effect. Some of these polysaccharides are not absorbed but stick to certain cells lining the gut and form a barrier preventing absorption of unwanted material so helping to prevent a "leaking" gut syndrome. In topical preparations the sugars are also the main moisturisers.
5. Anthraquinones
There are twelve of these Phenolic compounds which are found exclusively in the plant sap. In small quantities, when they do not exert their purgative effect, they aid absorption from the gastro-intestinal tract and have anti-microbial and pain killing effects. In some commercial health drinks, the anthraquinones are removed because of the fear of producing abdominal pain or diarrhoea, but I feel that they are actually beneficial in small amounts. The important ones, Aloin and Emodin, act as painkillers. They also function as anti-bacterials and anti-virals.
6. Lignins
This in itself is an inert substance but when included in topical preparations it endows Aloe Vera with a singular penetrative effect so the other ingredients are absorbed into the skin.
7. Saponins
These soapy substances form about 3% of the Aloe Vera gel and are capable of cleansing, having antiseptic properties. These act powerfully as anti-microbials against bacteria, viruses, fungi and yeasts.
8. Fatty Acids
Cholesterol, Campesterol, b. Sisosterol and Lupeol.
These four plant steroids are important anti-inflammatory agents.
9. Salicylic acid
An aspirin-like compound possessing anti-inflammatory and anti-bacterial properties.
10. Amino Acids
The body needs 22 amino acids – the gel provides 20 of these. More importantly, it provides 7 out of the 8 essential amino acids which the body cannot synthesise.

Properties of Aloe Barbadensis Constituents
There is a wide range of research from all over the world based upon different species of Aloe. As far as is possible the table below describes the constituents found in Aloe Barbadensis Miller (Aloe vera) with their properties and their inferred activities. The aloe constituents are derived from the aloe leaf which consists of three primary sections: the rind (photosynthesis) with sap contained in the pericyclic transport tubules (xylem and phloem), the mucilage (container) layer and the parenchyma or gel fillet (storage) layer. The relative quantities of the aloe constituents may be quite small (the total solid fraction varies from 0.5 to 1.5%), but most researchers claim that the effects that are observed may be due to the synergistic actions of the 75 known ingredients. The table below lists the constituents in alphabetical order. The aloe constitiuent list represents a consensus view of published work and may not be complete. The properties and the associated activities reported in the table have been constructed using the references cited below.

Constituents Number & Identification Properties & Activity Comment
Amino Acids Provides 20 of the 22 human required amino acids & 7 of the 8 essential ones Provides the basic building blocks of proteins in the production of muscle tissue etc The 8 essential amino acids are those the human body cannot manufacture
Anthraquinones Provides 12 anthraquinones: Aloe emodin, Aloetic Acid, Aloin, Anthracine, Antranol, Barbaloin, Chrysophanic Acid, Emodin, Ethereal Oil, Ester of Cinnamonic Acid, Isobarbaloin, Resistannol. In relatively small concentrations together with the Gel fraction they provide Analgesic, Antibacterial, Antifungal & Antiviral activity. In high concentration on their own they can be toxic. Traditionally known as laxatives.The antraquinones are found in the sap. The anthraquinone derivatives (anthrones & chromones) comprise the phenolic fraction of the sap. The primary sap component is Aloin/Barbaloin anthrone derivative
Enzymes Provides 8 enzymes: Aliiase, Alkaline Phosphatase, Amylase, Carboxypeptidase, Catalase, Cellulase, Lipase, Peroxidase Helps breakdown of food sugars and fats aiding digestion & enhancing nutrient absorbtion
Hormones Auxins & Gibberellins Wound Healing & Anti-inflammatory
Lignin Cellulose based substance Thought to provide penetrating power in Aloe vera skin preparations and may act as a carrier for other components
Minerals Provides 9 minerals: Calcium, Chromium, Copper, Iron, Magnesium, Manganese, Potassium, Sodium, Zinc Essential for good health and is known to work in certain combimation with each other, vitamins and other trace elements
Salicylic Acid Aspirin like compound Analgesic
Saponins Glycosides Soapy substance both cleansing and antiseptic
Sterols Provides 4 main plant steroids: Cholesterol, Campesterol, Lupeol, ß Sitosterol Anti-inflammatory agents. Lupeol also possesses antiseptic and analgesic properties
Sugars Monosaccharides: glucose & fructose
Polysaccarides: gluco-mannans / polymannose

Anti-inflammatory action
Anti-viral, immune modulating activity of Acemannan The long chain gluco-mannans are absorbed intact by the pinocytotic process of certain cells lining the digestive tract.
Vitamins A, C, E, B, Choline, B12, Folic Acid Antioxidant(A,C,E): neutralises free radicals B's & Choline involved in amino acid metabolism, B12 required for production of red blood cells, Folic Acid in the development of blood cells






Antimicrobial effect-
Aloe vera gel exhibits antifungal effects. In clinical studies, subjects with Candida albicans had a reduction in the number of yeast colonies in stool cultures, and a reduction in stool specific gravity (less dense.)
Aloe vera gel has demonstrated activity against bacteria in several studies. The antimicrobial effects against skin pathogens being slightly superior to silver sulfodiazine, a potent antiseptic used in the treatment of extensive burns.
A polysaccharide present in aloe called Acemannan has exciting antiviral and anti-retroviral and immunopotentiating effects such as inhibiting glycosylation of viral glycoproteins, enhancement of macrophage activity, immune system potentiators, T-cell function, and interferon production. Acemannan is now approved for veterinary use in fibrosarcomas and feline leukemia (a retro-virus), and in preliminary human and in vitro studies, as a synergistic enhancement to the drug azidothymidine (AZT) or Acyclovir to inhibit the replication of HIV and herpes simplex type 1 (HSV-1). Sarcoma research is also promising.
The anthroquinones and anthrones in the aloe latex probably produce their laxative effect by increasing colonic peristalsis and increasing the intestinal water content by opening chloride channels of the colonic membrane to cause a net reduction of liquid absorption by the colon ]. The anthroquinone glycosides reach the colon mostly undigested, although some are metabolized by enzymes produced by intestinal bacteria. The result includes more frequent stools with softer consistency. In most of the studies on the laxative effects of aloe, the aloe was not used alone but in combination with other laxatives, such as celandin or psyllium. Aloe's side effects can include abdominal pain, diarrhea, and electrolyte imbalances, especially at higher doses.
Species Identification For Aloe Barbadensis
Description and Tests.—ALOE BARBADENSIS (U. S. P.), Barbadoes aloes. This variety comes "in hard masses, orange-brown, opaque, translucent on the edges; fracture waxy or resinous, somewhat conchoidal; odor saffron-like; taste strongly bitter. Mixed with alcohol and examined under the microscope, it exhibits numerous crystals. Mixed with nitric acid, it acquires a red color. Barbadoes aloes is not colored, or acquires only a light bluish-green tint on being mixed with sulphuric acid and blowing the vapor of nitric acid over the mixture (difference from Natal aloes)"—(U.S. P.). Barbadoes aloes is not so bright and clear as the Socotrine variety, is of darker color, more compact texture, drier, though not so brittle, with a stronger and more disagreeable taste, being intensely bitter and nauseous, with little or nothing of the aromatic flavor of the Socotrine
Although Aloe vera is not indigenous to India and Chhattisgarh but as exotic herb the natives have welcomed and adopted this herb and this is the reason Aloe holds a reputed position as ethnomedicine. In Chhattisgarh, few decades back Aloe was present in every home garden and also grew naturally in dense forests but due to heavy demand of Aloe based herbal products its availability in natural habitat is decreasing rapidly and today it is present in isolated patches of Chhattisgarh.
Botanical differences among three major species of Aloe
Characteristics Aloe Aloe Aloe
barbadensis syn socotrina spicata
Aloe vera
1) Stem Woody, simple, cylindrical, short Woody, straight, 1.5 foot high and more, naked below, where it is stongly marked with the scars of leaves Stem 3-4 feet high, as thick as man's arm
2) Leaves Fleshy, amplexicaul, first spreading, then ascending, lanceolate, glaucous-green, flat above, convex below, armed with hard distant reddish spines perpendicular to the margin; a little mottled with darker colour, the paranchyma slightly coloured brown. Amplexicaul, ascending, ensiform, green, curved inwards at the point, convex below rather concave above, marked with numerous small white marginal serratures, the parenchyma abounding in a bright brownish-yellow juice Leaves thick, fleshy, broad at the base, gradually narrowing to the point, channelled, full 2 feet long, distantly toothed, with a few white spots, their parenchyma almost colourless.
3) Flowers Spike cylindrical ovate, flowers at first erect, then spreading, afterwards pendulous, yellow, not longer than the stamens Raceme cylindrical, unbranched, flowers scarlet at the base, pale in the middle, green at the point, stamens unequal, 3 of them longer than the flowers. Spike a foot long, very compact, with the flowers companulate and horizontal. The three petals broader, ovate, obtuse, white with a triple green like, the sepals narrower, less concave; stamens longer than the perianth, flowers are filled with honey
4) Origin The East Indies and Barbary Islands of Socotra Interior of the Cape of Good Hope
























Pseudomonas aeruginosa
Pseudomonas aeruginosa is the epitome of an opportunistic pathogen of humans. The bacterium almost never infects uncompromised tissues, yet there is hardly any tissue that it cannot infect if the tissue defenses are compromised in some manner.
Pseudomonas aeruginosa is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. The family includes other genera, which, together with certain other organisms, constitute the bacteria informally known as pseudomonads. These bacteria are common inhabitants of soil and water. They occur regularly on the surfaces of plants and occassionally on the surfaces of animals
Pseudomonas aeruginosa as pathogen Pseudomonas aeruginosa colonies on agar
Pseudomonas aeruginosa is an opportunistic pathogen, meaning that it exploits some break in the host defenses to initiate an infection. It causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns. The case fatality rate in these patients is 50 percent.
Pigment production by Pseudomonas aeruginosa
P. aeruginosa is capable of producing several pigments, of which the most characteristic is pyocyanin. As far as is known, this blue pigment is an absolute diagnostic character, since no other species has been found to produce it. Pyoverdin is the fluorescent pigment most often produced, but the bacteria may be able to produce several additional pigments, including a reddish pigment, pyorubrin, and a brown pigment, pyomelanin. This pigment, in common with other melanins, is produced from aromatic amino acids such as tyrosine or phenylalanine, while pyorubrin production is enhanced by the addition of glutamate to the medium. Besides pyoverdin, which acts as a siderophore, the function of these pigments is obscure.
Composition of King's Medium B. The mineral salts of this medium, as well as the exclusion of iron, enhance the production of pigments.In many laboratories, King's B Medium is used as a general medium for routine cultivation of Pseudomonas.
Proteose Peptone 2 g
Glycerol 1 g
K2HPO4 0.15 g
MgSO4•7H2O 0.15 g
Agar 1.5 g
Distilled water 100 ml
Adjust to pH 7.2




ISOLATION OF pseudomonas aeruginosa

Source: - Sewage and water and soil samples






Normal media Iron deficient media
(King’s B Media)




Iron present Iron absent




No pigments produced Production of pigments




No fluorescence produced
Under U.V





IDENTIFICATION OF pseudomonas aeruginosa

Grey regular colonies- found
Tends to spread on the agar surface- found
Pigment production (red fluorescence)- found
Flat colonies- found
Grows in iron deficient conditions- found
Gram staining Gram-negative

It is identified on the basis of its Gram morphology, inability to ferment lactose, a positive oxidase reaction, its fruity odor, and its ability to grow at 42° C . Fluorescence under ultraviolet light is helpful in early identification of P. aeruginosa colonies. Fluorescence is also used to suggest the presence of P. aeruginosa in wounds.

Pathogenesis of S. aureus infections
Staphylococcus aureus causes a variety of suppurative (pus-forming) infections and toxinoses in humans. It causes superficial skin lesions such as boils, styes and furunculosis; more serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections; and deep-seated infections, such as osteomyelitis and endocarditis. S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices. S. aureus causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by release of superantigens into the blood stream.




















Isolation of staphylococcus aureus




Source- swab from mouth and nose


Cotton sticks were used to collect the sample from mouth and nose.


Cotton sticks were swabbed on the surface of the solidified mannitol salt agar plates


The plates were incubated for three days at 35º C


MSA is a selective media for s. aureus, bacterial growth with the formation of aura confirms the presence of the concerned bacteria.




IDENTIFICATION OF S. aureus

Grows in high salt conc. 75gms/1000 ml (halophilic bacteria)- found
Forms yellow halo/aura on MSA- found
Forms isolated round colonies found
Gram staining Gram-positive


catalase positive
coagulase positive
golden yellow colony on agar
normal flora of humans found on nasal passages, skin and mucous membranes
pathogen of humans, causes a wide range of suppurative infections, as well as food poisoning and toxic shock syndrome












Coliforms

Coliforms are a broad class of bacteria found in our environment, including the feces of man and other warm-blooded animals. The presence of coliform bacteria in drinking water may indicate a possible presence of harmful, disease-causing organisms.
The coliform group includes species from the genera Citrobacter, Klebsiella, Enterobacter, and E.coli. Other than E. coli, no foodborne outbreaks have been associated with coliforms. Coliforms were historically used as "indicator microorganisms" to serve as a measure of fecal contamination, and thus potentially of the presence of enteric pathogens. Although some coliforms are found in the intestinal tract of man, most are found throughout the environment and have little sanitary significance.
A wide groups of coliforms are also responsible for food spoilage, causing degradation of processed and unprocessed food which in turn becames harmful for the human beings.
Isolation of coliform-
coliform group of bacteria is present all around In water, soil sources, thus this group of bacteria can easily be isolated from a polluted sample of water.
1. Polluted water samples were collected
2. Brilliant Green Bile Broth was prepared (100 ml), poured in test tubes with varying amount as shown in the table.

3. Five replicates of each of the tube type was taken, as shown in the table

4. Durham’s tube was inserted into each tube

5. 2 ml of the Sample was diluted with 10 ml of distilled water.

6. .2 ml of this solution was added in each tube containing broth.

7. Inoculated for 24 hrs at 35C.

8. Production of gas, turbidity and disappearance of green color confirms the presence of coliforms in the tubes




Culture medium: Use brilliant green lactose bile broth fermentation tubes for the confirmed phase.
Brilliant green lactose bile broth:

Formula Per Liter
Peptone ................................................................... 10.0 g
Oxgall ....................................................................... 20.0 g
Lactose ..................................................................... 10.0 g
Brilliant Green .......................................................... 13.3 mg


Identification of Coliforms
Brilliant green colour of the broth changed to turbid found
Accumulation of gas in Durham’s tube after 24 hrs incubation found
Gram test found gram negative
Isolating E. coli on EMB showed metallic shine






































For testing antimicrobial susceptibily of aloe vera against various pathogenic micro organism, two types of extract were prepared . These two extract were

1. Aloe vera gel extract
2. aloe vera leaf extract

PREPARATION OF ALOE VERA GEL EXTRACT

Healthy thick leaves were taken


100 gms of gel was drawn out


Gel was crushed to liquid


Total volume was made to 200ml


Thus, 50% w/v of gel was prepared


Shaken with orbital shaking incubator at slow rpm for 15 min. at room temperature


** The gel has to be freshly prepared to use because it shows brown apple syndrome i.e. browning of the gel extract within few hours of extraction due to oxidation of some of its components.



For coliforms- 50% (W/V) gel
Pseudomonas aeruginosa- 50% (W/V) gel
Staphylococcus aereus- 50% (W/V) gel
5 ml of the above conc. was diluted with 10 ml distilled water












PREPARATION OF ALOE VERA LEAF EXTRACT




The leaves remaining after drawing off the gel were washed



20 gms was weighed



Left for 3 days for air drying and crushed



Soaked in 30 ml of ethanol for four days



This was then filtered with filter paper and filtrate was obtained



The filtered was concentrated to solid crystals by slow evaporation of alcohol and water



The solid crystals were then dissolved in 15 ml of distilled water



Stored chilled till use















AIM-

Spectrophotometrical analysis of antimicrobial susceptibility testing of Aloe vera gel against Pseudomonas aeruginosa.

REQUIREMENTS-

Broth- King’s B Broth
Gel- 50% (W/V) in distilled water. (5 ml diluted with 10 ml distilled water)
Gel Conc. - 0,.5,1.0, 1.5, 2.0, 2.5.
Incubation- 37º C for 24 hrs
Pure culture of Psuedomonas aeruginosa

PROCEDURE-

1. King’s B Broth was prepared (100 ml), poured in test tubes with varying amount as shown in the table.
2. Five replicates of each of the tube type was taken, as shown in the table

3. Loop full of culture was taken from the pure culture plates of Pseudomonas aeruginosa, Dissolved in 10 ml of water.

4. .2 ml of this solution was added in each tube containing broth.

5. Inoculated for 24 hrs at 37 C

6. Readings were taken in spectrophotometer without adding the gel

7. Gel was added in the tubes, with volumes as shown in the table to make the total volume to be 4 ml

8. Further readings were taken with a time interval of 30 min. incubation.

9. Data were recorded and graph was plotted as absorption vs time


RESULT- The aloe vera gel extract was found out to have no significant effect on
Pseudomonas aeruginosa, as none of the experimental gel concentration was found
to pose a growth retarding effect on the bacteria.




** Reference was taken as uninoculated broth.
Readings were taken at 600nm.





Discussion:-
The aloe vera gel has not been found to have any significant effect on Pseudomonas aeruginosa. The optical density for the broth of these bacteria with all experimental gel concentration was found to be increasing. This increase is like the elevation found in the graph of the control, having no gel at all. Amount of bacteria was found to be increasing with time. No depression in curve was found at any time. This shows that with the increase optical density the bacterial concentration was increasing with respect to time







AIM-

Spectrophotometrical analysis of antimicrobial susceptibility testing of Aloe vera gel against coliforms.

REQUIREMENTS-

Broth- Brilliant Green Bile Broth
Gel- 50% (W/V) in distilled water. (5 ml diluted with 10 ml distilled water)
Gel Conc. - 0,.5,1.0, 1.5, 2.0, 2.5 ml.
Incubation- 35 C for 24 hrs
Sample- sewage water Brilliant Green Bile Broth

PROCEDURE-

1. Brilliant Green Bile Broth was prepared (100 ml), poured in test tubes with varying amount as shown in the table.

2. Five replicates of each of the tube type was taken, as shown in the table

3. Durham’s tube was inserted into each tube

4. 2 ml of the Sample was diluted with 10 ml of distilled water.

5. .2 ml of this solution was added in each tube containing broth.

6. Inoculated for 24 hrs at 35C.

7. Production of gas, turbidity and disappearance of green color confirms the presence of coliforms in the tubes

8. Readings were taken in spectrophotometer without adding the gel

9. Gel was added in the tubes, with volumes as shown in the table to make the total volume to be 4 ml

10. Further readings were taken with a time interval of 30 min. incubation.

11. Data were recorded and graph was plotted as absorption vs time


RESULT- The aloe vera gel extract was found out to have significant effect on coliforms.
With an increase in the gel concentration the effect was found to be increasing. At
the same growth in the control tube was rapidly found to be increasing.

Discussion:-
The curve plotted to show the effect of antimicrobial effect of aloe vera gel on coliforms shows that, the gel has got a significant effect on coliforms. The graph plotted for the same shows that every curve (for each gel concentration) except of the control goes down from their origin point. Thus this can be concluded that the optical density for all the gel concentration is decreasing, it thus indicates that the concentration of the organism is either decreasing or at least has became stable. On the other hand, for the control in which no gel has been added the curve goes up with respect to the time. The difference between the curves of control sample and the samples with gel, clearly shows that gel has got considerably significant
amount of effect on coliforms. From the graph it can also be concluded that the effect also increases with the increasing concentration of the gel. More the gel the time of action that much goes less.



** Reference was taken as uninoculated broth.
Readings were taken at 660nm.

AIM-

Spectrophotometrical analysis of antimicrobial susceptibility testing of Aloe vera gel against Staphylococcus aureus

REQUIREMENTS-

Broth- Mannitol Salt Broth & Nutrient broth
Gel- 50% (W/V) in distilled water. (5 ml diluted with 10 ml distilled water)
Gel Conc. - 0, .5,1.0, 1.5, 2.0, 2.5.
Incubation- 37º C for 24 hrs
Pure culture of Staphylococcus aureus


PROCEDURE-

1. Mannitol Salt Broth & Nutrient broth was prepared (100 ml), poured in test tubes with varying amount as shown in the table.

2. Five replicates of each of the tube type was taken, as shown in the table

3. Loop full of culture was taken from the pure culture plates of staphylococcus aureus, Dissolved in 10 ml of water.

4. .2 ml of this solution was added in each tube containing broth.

5. Inoculated for 24 hrs at 35C.

6. Readings were taken in spectrophotometer without adding the gel

7. Gel was added in the tubes, with volumes as shown in the table to make the total volume to be 4 ml

8. Further readings were taken with a time interval of 30 min. incubation.

9. Data were recorded and graph was plotted as absorption vs time


RESULT- In the graph plotted, the curve for the control increases with time, while for other gel concentrations the curve is either stabilized or goes downwards from the point of origin. Which shows that the gel has got a significant effect on the bacteria.

Discussion:-
The curve obtained shows that without any gel the bacteria tends to increase, but with different gel concentrations, the growth stabilizes and bacterial concentrations in the sample solutions then decreases. The decrease in the optical density with time shows that bacterial concentration is decreasing in sample solution with respect to increase in time. This shows that aloe vera gel has got a significant antibacterial action on Staphylococcus aureus. The stabilization or decrease in the optical density is due to the stopped bacterial growth and hence turbidity.




** Reference was taken as uninoculated broth.
Readings were taken at 600nm for nutrient broth





p. aeruginosa

Tube

King’s B Broth
(ml)
Vol. of inoculum (ml)
Vol. of gel
(ml)
Total Vol.
(ml)
Gel free x 5 3.8 0.2 0 4
0.5 x 5 3.3 0.2 0.5 4
1.0 x 5 2.8 0.2 1.0 4
1.5 x 5 2.3 0.2 1.5 4
2.0 x 5 1.8 0.2 2.0 4
2.5 x 5 1.3 0.2 2.5 4

S.aureus
Tube
MS & Nutrient Broth
(ml)
Vol. of inoculum (ml)
Vol. of gel
(ml)
Total Vol.
(ml)
Gel free x 5 3.8 0.2 0 4
0.5 x 5 3.3 0.2 0.5 4
1.0 x 5 2.8 0.2 1.0 4
1.5 x 5 2.3 0.2 1.5 4
2.0 x 5 1.8 0.2 2.0 4
2.5 x 5 1.3 0.2 2.5 4

Coliforms
Tube

BGBB
(ml)
Vol. of inoculum (ml)
Vol. of gel
(ml)
Total Vol.
(ml)
Gel free x 5 3.8 0.2 0 4
0.5 x 5 3.3 0.2 0.5 4
1.0 x 5 2.8 0.2 1.0 4
1.5 x 5 2.3 0.2 1.5 4
2.0 x 5 1.8 0.2 2.0 4
2.5 x 5 1.3 0.2 2.5 4






AIM: - Determination of antimicrobial activity of aloe vera leaf extract on pseudomonas aeruginosa.

PRINCIPLE:-
The leaf extracts from aloe vera contains various dissolved compounds among which some exibit antibacterial effect namely anthraquinone phenol derivative emoin ,aloe-emodin.these compounds possess an growth inhibitory action on pseudomonas aeruginosa .the formation of zone of inhibition around the wells containing the leaf extract will depict the inhibitory action of leaf extract on the concerned bacteria.

REQUIREMENTS:-
24 hours old culture of pseudomonas aeruginosa
Cork borer,leaf extract ,nutrient agar medium

PROTOCOL:-
1. Bacterial suspension from the pure culture of pseudomonas aeruginosa was made.
2. Nutrient agar medium was prepared.
3. 1 ml of bacterial suspension was poured into the sterilized plate onto which cooled molten nutrient medium was poured and was left solidification.
4.Wells were made (4 wells per plate) on the solidified agar with the help of cork borer(4mm in diameter).
5.0.1 ml leaf extract was poured into well with the help of 1ml sterilized pipette.
6.Plate were incubated at 370C for 48 hours.
7. After two days incubation zone of inhibition was observed and the diameter was measured for each of the plate.
8. For each plate mean of all the 4 zone of inhibition was calculated. This mean was the final diameter for each zone of inhibition.

RESULTS: - The zone of inhibition was obtained around the wells. The mean diameter for the various zone of inhibition formed in the wells were found to be as follows:-
14 mm 18 mm
18 mm 18 mm
16 mm 16 mm
14 mm 15 mm
Overall diameter = 16.12 mm


DISCUSSION: - The zone of inhibition around the wells containing the leaf extract was found. The zone was clearly visible around the wells. The zones were having significant magnitude in diameter. This shows that aloe vera leaf extract has got an considerable amount of antibacterial effect on pseudomonas aeruginosa.



AIM: - Determination of antibacterial activity aloe vera gel extract on Pseudomonas aeruginosa

PRINCIPLE:-
The gel extracts from aloe vera contains various dissolved compounds among which some exhibit antibacterial effect namely anthraquinone phenol derivative emoin ,aloe-emodin.these compounds possess an growth inhibitory action on Pseudomonas aeruginosa .the formation of zone of inhibition around the wells containing the gel extract will depict the inhibitory action of leaf extract on the concerned bacteria.

REQUIREMENTS:-
24 hours old culture of Pseudomonas aeruginosa
Cork borer, gel extract , nutrient agar medium

PROTOCOL:-
1. Bacterial suspension from the pure culture of Pseudomonas aeruginosa was made.
2. Nutrient agar medium was prepared.
3. 1 ml of bacterial suspension was poured into the sterilized plate onto which cooled molten nutrient medium was poured and was left solidification.
4. Wells were made (4 wells per plate) on the solidified agar with the help of cork borer(4mm in diameter).
5.0.1 ml gel extract was poured into well with the help of 1ml sterilized pipette.
6. Plate were incubated at 370C for 48 hours.
7. After three days incubation zone of inhibition was observed.


RESULTS: - The zone of inhibition was not obtained around the wells. This shows that gel extract has no antibacterial effect on
Pseudomonas aeruginosa.

DISCUSSION:- The zone of inhibition around the wells containing the gel were not formed .The bacterial growth was even found over the wells which shows that aloe vera gel has no effect on the growth of seudomonas aeruginosa.

AIM: - Determination of aloe vera leaf extract on Aspergillus fumigatus.

PRINCIPLE:-
The leaf extracts from aloe vera contains various dissolved compounds among which some exhibit antifungal effect namely anthraquinone phenol derivative emoin ,aloe-emodin.These compounds possess an growth inhibitory action on various fungal species namely Candida albicans .The fungal effect of aloe vera leaf could be tested against Aspergillus fumigatus by formation of zone of inhibition. The formation of zone of inhibition around the wells containing the leaf extract will depict the inhibitory action of leaf extract on the concerned fungi.

REQUIREMENTS:-
24 hours old culture of Aspergillus fumigatus
Cork borer, leaf extract, Cpazek dox medium

PROTOCOL:-
1. Cpazek dox medium was prepared and was autoclaved.
2. Cooled C-dox medium was poured into the sterilized plate and was left solidification.
4. Wells were made (4 wells per plate) on the solidified agar with the help of cork borer (4mm in diameter).
5.0.1 ml leaf extract was poured into well with the help of 1ml sterilized pipette.
6. Point inoculation of Aspergillus fumigatus culture was done.
7. Plate were incubated at 260C for 3 days
8. After three days of incubation zone of inhibition was observed.


RESULTS: - The zone of inhibition was obtained around the wells. This shows that gel has antifungal effect on
Aspergillus fumigatus. The mean diameter for each of the inhibiton zone are as follows:-
10 mm 12 mm
8 mm 6 mm
8 mm 9 mm
5 mm N.A
Overall mean diameter = 8.2 mm

DISCUSSION: - The zone of inhibition around the wells containing the leaf extract was found. The zone was clearly visible around the wells. The zones were having significant magnitude in diameter. This shows that aloe vera leaf extract has got a considerable amount of antifungal effect on Aspergillus fumigatus AIM: - Determination of aloe vera gel extract on Aspergillus fumigatus.

PRINCIPLE:-
The gel extracts from aloe vera contains various dissolved compounds among which some exhibit antifungal effect namely anthraquinone phenol derivative emoin ,aloe-emodin.These compounds possess an growth inhibitory action on various fungal species namely Candida albicans .The fungal effect of aloe vera gel could be tested against Aspergillus fumigatus by formation of zone of inhibition. The formation of zone of inhibition around the wells containing the gel extract will depict the inhibitory action of gel extract on the concerned fungi.

REQUIREMENTS:-
24 hours old culture of Aspergillus fumigatus
Cork borer, gel extract ,Cpazek dox medium

PROTOCOL:-
1. Cpazek dox medium was prepared and was autoclaved.
2. Cooled C-dox medium was poured into the sterilized plate and was left solidification.
4. Wells were made (4 wells per plate) on the solidified agar with the help of cork borer(4mm in diameter).
5.0.1 ml gel extract was poured into well with the help of 1ml sterilized pipette.
6. Point inoculation of Aspergillus fumigatus culture was done.
7.Plate were incubated at 260C for 3 days
8. After three days of incubation zone of inhibition was observed.


RESULTS: - The zone of inhibition was not obtained around the wells. This shows that gel has no antifungal effect on
Aspergillus fumigatus

DISCUSSION: - The zone of inhibition around the wells containing the gel were not formed .The fungal growth was even found over the wells which shows that aloe vera gel has no effect on the growth of Aspergillus fumigatus















Keratinophilic Fungi

The biggest group of organisms that can utilize keratin as the sole source of carbon and nitrogen are the keratinophilic fungi.These are minute organisms that cannot be seen by the naked eye, unlike macrofungi like mushrooms. The word keratinophilic means ‘keratin loving’, can utilize keratin as the sole source of carbon and nitrogen are the
keratinophilic fungi.

Keratinophilic fungi mainly effect the outer parts in humans, aloe vera being effective against many outer harms as like healing wounds, skin and hair problems etc, it is worthy to test the effect of aloe vera extracts on the keratinophilic fungi. Aloe vera extracts may not be effective against all the keratiniphillic fungi, but it may have retarding effect or possibly may not have any effect on some species of the specified organisms. Aloe vera being used as a traditional healer in skin and hair problems could be sffective against some species of keratinophilic fungi, its extracts contains a large number of such compounds which are loudly effective against a number of bactera and fungi.

Keratinophilic fungi can be isolated on a media containing keratin powder as the only carbon source in a media suitable for the growth of fungi. If the such a fungi exists in the media which can utilizing the keratin powder could be considered as the keratin degrading or as keratinophilic fungi.

The susceptibility of the keratinophilic fungi against the can be simply tested by the media-wells method. A suitable media facilitating the growth of fungi can be used. Wells were made into which aloe vera extracts were poured. The plates were left for incubation. Formation of zone of inhibition around the wells shows that the inoculated species of fungi has an susceptibility against the aloe vera extract
























AIM-

Evaluation of the antimicrobial activity of the aloe vera extracts on keratinophilic fungi


Materials-
Two samples of keratiniphillic fungi
culture media- czapecks dox media pH- 4.3
Gel conc.- 50 % (W/V) 5 ml diluted with 10 ml distilled water
Leaf conc.- extract made with 20 gms wet weight of aloe vera leaf
Cork borer



Procedure-
1. Molten culture media was poured into the sterile culture, plates left for solidification.
2. Flamed cork borer was used to make the wells in the solidified media
3. Eight wells per plates were made
4. In half of the plates marked G .1 ml of gel was poured into each well using sterile pipette.
5. In the other half plates marked L.1 ml of leaf extract was poured into each well.
6. Keratinophilic fungi were inoculated on solidified media by point inoculation
7. The inoculated plates were incubated at 260 C for 5 days
8. The zone of inhibition formation was studied on the incubated plates.
9. Three measurements of each zone of inhibition was made, their mean was calculated and recorded.




Result – Out of two Keratinophillic fungi, tested for the susceptibility, one from the fish scale sample formed zone of inhibition where the well contained leaf extract from the aloe vera. The mean diameters of the zone of inhibition are as follows:-

15 mm 10mm
12mm 10mm
10mm 11mm
8mm 7mm



Discussion:-
Keratinophillic fungi infect mainly the skin, nails, hairs etc. With this idea we estimated that and aloe vera leaf extract being effective against many of the skin disease causing bacteria and fungi. It may have an inhibitory action on the keratinophillic fungi.
It may not be having activity against all the keratinophillic fungi, but out of two species we tested, one showed susceptibility against only the leaf extract.











Microscopic examination of the effect of Aloe vera gel extract on bacterial motility/activity.

Bacteria show motility under microscope when viewed with the hanging drop slide preparation. Bacteria shoes two types of movement when viewed under microscope
1- Actual motility of the bacteria.
2- Brownian movement due to collision with the water molecules.

Decreased or stopped bacterial movement could be a sign of retardation in the bacterial activity. Motile bacteria will show its natural motility under the microscope when not treated with any substance which can hinder the bacterial growth, the bacterial growth, in other words its motility will get stopped or retarded.

Application of such a substance having antimicrobial activity could be risky form them for which they show retarded growth and motility.

Microscopic examination of the effect of Aloe vera gel extract on bacterial motility/activity

Coliforms
Staphylococcus aureus
Pseudomonas aeruginosa

Requirements: -
12 hrs old broth culture
Hanging drop( cavity) slide
Cover slip
Glycerin

Procedure: -
1. Bacterial suspension was made with different gel concentration along with without gel bacterial suspension was also made.
2. Hanging drop slide was cleaned and flamed and was placed on the table with dipresssion uppermost.
3. A little glycerine was spreaded around the cavity of the slide
4. Cover slip was cleaned and was applied on each of the four corners of the cover slip using the needle.
5. The cover slip was placed on a clean paper with glycerin side up.
6. One loopful of culture was transferred in the centre of the cover slip.
7. The depression slide was placed onto the coverslip, with the cavity facing down so that covers the suspension.
8. The slide was gently pressed to form a seal between the cover slip and the
Slide.
9. The prepration was lifted & was quickly turned the hanging drop prepration coverslip up so that the culture drop is suspended.
10. The prepration was examined under 100x power objective with reduced light.
11. The time of motility retardation of different each gel concentration was noted.

Observations: -
The bacterial motility was observed with the normal slides, but retardation was found and ultimately motility stopped in case of coliforms and Staphylococcus aureus. But Pseudomonas aeruginosa showed no result towards the aloe vera gel of any concentration.

Observation Table



Bacteria


Gel conc.
(ml)
Mean
Time of motility stoppage (min.)



Coliforms






0x3

Motility continued

0.5x3
No clear result
1.0x3

11
1.5x3
10

2.0x3
7

2.5x3
5



Staphylococcus
aureus




0x3

Motility continued
0.5x3

No clear result
1.0x3

No clear result
1.5x3 11
2.0x3
10
2.5x3 8


Pseudomonas aeruginosa 0x3 Motility continued
0.5x3 Motility continued
1.0x3 Motility continued
1.5x3 Motility continued
2.0x3
Motility continued
2.5x3 Motility continued

Result:-
The aloe vera gel posed a significant antimicrobial effect as the bacterial motility stopped in after some time as in case of Coliforms and Staphylococcus aureus. But the viability of Pseudomonas areuginosa continued beyond the experimental time (15-20 min.)

Discussion:-
When the normal bacterial suspension was viewed with hanging drop technique under the microscope showed considerable motility, which also showed their viability. With difertent gel concentrations dissolved to the suspension, the motility or the viability of the bacteria stopped after some time. This shows that gel has got an antimicrobial action on the two bacteria out of three. In case of coliforms the time of action shows a trend which shows that with an increase in the gel concentration the time of action of the gel gradually decreased.
Whereas in case of pseudomonas aeruginosa the motility continued even after the experimental time limit.





Hanging Drop Technique

































































To determine the phenol coefficient of Aloevera Gel

Principle: -

The phenol coefficient is a comparative test to determine the effectiveness of various phenolic based chemicals which is compared to phenol under standardized experimental condition. The phenol coefficient test is limited to bactericidal & phenol like compounds & cannot be used to evaluate bacteriostatic compounds. Phenol & its derivatives called phenolics kill bacteria by damaging the plasma membrane, inactivating enzymes & denaturing proteins. The aloevera gel contains phenolic compounds called anthraquinone which are bactericidal in nature.

This exercise deals with comparing the effectiveness of anthraquinones & finding out its phenol coefficient.

Requirements: -

24 hours nutrient broth culture of staphylococcus aureus.
Nutrient broth tubes (19).
Phenol dilution : 1:80, 1:90, 1:100.
Aloevera gel dilution : 1:70,1:80, 1:90.
I ml sterile pipette.
Test tube rack.

Procedure: -

1) 1 test tube each of different phenol & aloevera gel dilution was placed in a test tube rack.
2) With a sterile 1 ml pipette, rapidly 1 drop of the bacterial culture was added in ot each of the test tube.
3) The time of introduction into the test tubes was recorded.
4) The contents of the tubes was mixed.
5) Aseptically at intervals of 5, 10 & 15 minutes, 1 loopful from each of the test tubes {containing the phenolic compounds + S. aureus} was transferred into the appropriately labelled nutrient broth tubes.
6) 1 sterile test tube of nutrient broth was kept as control.
7) All the nutrient broth culture was incubated at 37oc for 48 hours.
Observation: -

Observe all the incubated broth cultures of S. aureus for the presence of growth in two different phenolics.

Results: -
Record the presence (+) or absence (-) of growth of the microorganisms in subcultures after an interval of 5, 10 & 15 minutes in a tabular form as given below:

The dilution of the test substance (i.e. aloevera gel) that killed the bacteria in 10 minutes but not in 5 minutes was circled. Again, the highest dilution of phenol that killed the bacteria in 10 minutes, but not in 5 minutes was circled.

No significant phenol coefficient of the aloe vera gel was found out, It shows hat its effectiveness is not as faster as that of the phenol




Calculation: -

The phenol coefficient of the test chemical was calculated as follows: -

Phenol coefficient = Reciprocal of the test chemical dilution circled
of the test chemical Reciprocal of the phenol dilution circled

A phenol coefficient greater than 1 suggest that the test chemical is more effective than phenol

Discussion:-.
The phenol coefficient of the aloe vera gel has not been significantly found out. Phenol is a potent disinfectant, having a faster action on the microbes. The gel contained many dissolved substances; in the previous experiments it has been found that gel has activity against the Staphylococcus aureus. Thus the trial shows that it is not as effective as like the phenol