With food and air- borne infections rising by the day, skin infections are the most commonly known infections. Staphylococcus aureus bacteria generally cause skin infections that lead to various diseases. Antiseptics are well known for preventing the bacteria from infecting the skin. Therefore in my experiment I would like to conduct a check on “The effect of three antiseptics on Staphylococcus aureus.”, keeping the time for the antiseptic to act on the surface constant but varying the dilutions to check the effect on the rate of bacterial growth on the surface. This experiment has been proved successful and is termed as the microbial challenge test.
Antiseptics and disinfectants are used extensively in hospitals and other health care settings for a variety of topical and hard-surface applications. In particular, they are an essential part of infection control practices and aid in the prevention of nosocomial infections. A wide variety of active chemical agents (or “biocides”) are found in these products, many of which have been used for hundreds of years for antisepsis, disinfection, and preservation. “Biocide” is a general term describing a chemical agent, usually broad spectrum that inactivates microorganisms. Because biocides range in antimicrobial activity, other terms may be more specific, including “-static,” referring to agents which inhibit growth (e.g., bacteriostatic, fungistatic, and sporistatic) and “-cidal,” referring to agents which kill the target organism (e.g., sporicidal, virucidal, and bactericidal).
Types of Antiseptics
Chlorhexidine Gluconate
A biguanidine derivative, used in concentrations of 0.5–4.0% alone or in lower concentrations in combination with other compounds, such as alcohols. Used as a skin antiseptic and to treat inflammation of the gums (gingivitis). The microbicidal action is somewhat slow, but remanent. It is a cationic surfactant, similar to Quats.
Alcohols
Most commonly used are ethanol (60–90%), 1-propanol (60–70%) and 2-propanol/isopropanol (70–80%) or mixtures of these alcohols. They are commonly referred to as "surgical alcohol". Used to disinfect the skin before injections are given, often along with iodine (tincture of iodine) or some cationic surfactants (benzalkonium chloride 0.05–0.5%, chlorhexidine 0.2–4.0% or octenidine dihydrochloride 0.1–2.0%).
Quaternary ammonium compounds
Also known as Quats or QAC's, include the chemicals benzalkonium chloride (BAC), cetyl trimethylammonium bromide (CTMB), cetylpyridinium chloride (Cetrim, CPC) and benzethonium chloride (BZT). Benzalkonium chloride is used in some pre-operative skin disinfectants (conc. 0.05–0.5%) and antiseptic towels. The antimicrobial activity of Quats is inactivated by anionic surfactants, include chlorhexidine andoctenidine.
Infection: Before a microbe or parasite can invade the host and cause infection, it must first attach to and penetrate the surface of the epithelial layers of the body. Organisms gain entrance into the body through active or passive means. That is they have to pass physical barriers such as the skin or epithelial cells which line the mucosal surfaces of the respiratory, gastro intestinal and genitourinary tracts. The active passage for these microbes and parasites are the dead layers of the skin.
Skin: The term ‘skin’ applies to the outer covering of vertebrate animals. The skin is the largest organ of the body and has many different functions. The structure of the skin varies in different vertebrate groups. The basic structure of human skin will be described here. The skin is composed of two main layers, the epidermis and dermis. These cover the underlying, or ‘subcutaneous’ tissue, which contains specialised fat- containing cell known as adipose tissue. The thickness of the epidermis and dermis varies according to the region of the body and from person to person.
Epidermis- The cells of this region are separated from the dermis by a basement membrane. The epidermis is composed of many layers of cells that form a stratified epithelium. The cells immediately above the basement membrane are cuboidal epithelial cells and form a region known as the Malpighian layer. The cells in this layer are constantly dividing by mitosis and replace all the cells of the epidermis as they wear away. The Malpighian layer forms the lower region of the stratum granulosum, which is composed of living cells that become flatter as they approach the outer region of the epidermis, the stratum corneum. Cells in this region become progressively flattened and synthesise keratin, which is a fibrous protein which makes the cells waterproof. As the keratin content of the cells increases, they are said to become ‘cornified’. Their nuclei disappear and the cells die. The thickness of the stratum corneum increases in part of the body where there is considerable friction, such as the ball of the foot and the bases of the fingers. The outer covering of the skin forms a semi- transparent, thin, tough, flexible, waterproof covering by the hair follicles and by pores, which are the pores, which are the openings of the sweat glands. The outermost squamos epithelial cells are continually being shed as a result of friction.
The stratum corneum has become modified in many vertebrates to produce nails, claws, hooves, horns, antlers, scales, feathers and hair. Keratin is the main component of all these structures.
Dermis- The dermis is a dense matrix composed of connective tissue rich in elastic fibres and contains blood capillaries, lymph vessels, muscle fibres, sensory cells, nerve fibres, pigment cells, sweat glands and hair follicles.
The skin also has other more specific immune functions. Langerhan cells, which are immunocompetent cells, are involved in recognizing and processing foreign substances. These decrease with age, altering immune function (Gilchrest, 1999).
Mucous membranes, covering our eyes, alimentary canal, and genito-urinary tracts, play a protective role in providing a barrier and differential absorption. Usually, the skin prevents invasion by microorganisms unless it is damaged—for example, by an injury, insect bite, or burn.
Staphylococcus Aureus: Out of the many bacteria entering the human body through the dead skin, Staphylococcus aureus, is the most potent organism infecting the skin.
Facultatively anaerobic - do not require oxygen for growth but do grow better in its presence
The most common species of staphylococcus is Staphylococcus aureus which is a pathogen of both humans and animals. It is characterized by its ability to clot blood plasma by action of the enzyme coagulase
a) Colony Characteristics of Staphylococcus aureus
Size - 1 micrometre
Gram nature - Gram positive
Appearance- Grape-like cluster but some occur as single or pair of cells
usually non-capsulate
Form - Colonies are circular, 2-3 nm in diameter with a smooth shiny surface when grown on nutrient agar, milk agar or blood agar for 24 h at 37 degrees
Pigmentation - Colonies are often pigmented, though a few strains are unpigmented
Halophile i.e.Salt-tolerant
b) Pathogenesis of Staphylococcus aureus
Present in the nose of 30% of healthy people and may be found on the skin, causes infection at sites of lowered host resistance.
Eg: - damaged skin or mucous membranes
c) Epidemiology of Staphylococcus aureus
i) Infected Lesions:
Large no. of staphylococci are disseminated (spread widely) in pus and dried exudates discharged from large infected wounds, burns, secondary infected skin lesions and in sputum coughed from the lung of a patient with bronchopneumonia. Direct contact is the most important mode of spread, but air-borne dissemination may also occur
ii) Healthy Carriers:
Spread into environment by the hands, handkerchiefs, clothing and dust consisting of skin squames? and cloth fibres of healthy carriers. Some carriers, called shedders, disseminate exceptionally large no. of cocci, comparable to the number disseminated by patients with large superficial lesions or lower respiratory tract infections
iii) Animals:
Domesticated and some wild species may disseminate Staphylococcus aureus from infected lesions or carriage sites and so cause infections in humans.
Mode of infection may be either exogenous (from an external source) or endogenous (from a carriage site or minor lesion, elsewhere in the patient's own body). Staphylococci do not grow outside the body except occasionally in moist nutrient materials such as meat, milk and dirty water. Although not spore-forming, they may remain alive in a dormant state for several months when dried in pus, sputum, bed clothes or dust
They are fairly readily killed by heat (65 degrees for 30 min), by exposure to light and by common antiseptics. Therefore I would like to study on the research question,
Resistance to antimicrobial agents (AMR) has resulted in morbidity and mortality from treatment failures and increased health care costs. Appropriate antimicrobial antiseptic use has unquestionable benefit, but physicians and the public frequently use these agents inappropriately.
The microbial challenge test would be most appropriate to study the effect of antiseptics on Staphylococcus bacteria.
Microbiological challenge testing has been and continues to be a useful tool for determining the ability of a product to prevent the growth of pathogens. Microbiological challenge tests also play an important role in the validation of processes that are intended to deliver some degree of lethality against a target organism or group of target organisms.
The design, implementation, and assessment of microbiological challenge studies is a complex task that depends on factors related to how the product is formulated, manufactured, packaged, distributed, prepared, and used.
When conducting a microbiological challenge study, a number of factors must be considered.
These include:
(1) the selection of appropriate pathogens,
(2) the level of challenge inoculum,
(3) the inoculum preparation and method of inoculation,
(4) the duration of the study,
(5) formulation factors and storage conditions,
(6) sample analyses.
The interpretation of the data and pass/fail criteria are critical in evaluating whether a product needs time/temperature control for safety.
It is also important to incubate and prepare the challenge suspension under standardized conditions and format. Shifts in the incubation temperature used to propagate the challenge organisms and the storage temperature of the product have been shown to change the length of the lag period of the challenge study itself.
The inoculum level used in the microbiological challenge study depends on whether the objective of the study is to determine product stability and shelf life or to validate a step in the process designed to reduce microbial numbers.
The preparation of the inoculum to be used in microbiological challenge testing is an important component of the overall protocol. Typically, for vegetative cells, 18 to 24 h cultures revived from refrigerated broth cultures or slants or from cultures frozen in glycerol are used. The challenge cultures should be grown in media and under conditions suitable for optimal growth of the specific challenge culture.
The method of inoculation is another extremely important consideration when conducting a microbiological challenge study. Every effort must be made not to change the critical parameters of the product formulation undergoing challenge. There are a variety of inoculation methods that can be used depending upon the type of product being challenged.
Standard suspensions of insoluble Barium sulphate precipitates have been described by Mc. Farland (1907) and Brown (1919-20). A series of standards of different BaSO4 concentrations correspond to suspensions of different numbers of bacteria/ml.
Preparation of Mc. Farland standards.
Std. Tube no.
BaCl2 1% (ml)
H2SO4 1% (ml)
Corresponding bacterial concentrations (millions/ml)
0.5
0.05
9.95
150
1
0.1
9.9
300
2
0.2
9.8
600
3
0.3
9.7
900
4
0.4
9.6
1200
5
0.5
9.5
1500
Use:
In a tube of the same internal diameter as the standard, prepare a uniform suspension in saline of bacteria under test to a density greater than that required.
Select a standard opacity tube of the density required and shake it vigorously until all the deposit is raised into uniform suspension.
At once compare the bacterial suspension with the standard. View with oblique illumination against a dark background or over a printed page.
Adjust the bacterial suspension by diluting with saline until it matches the standard, re- shaking the standard before each comparison.
Total Viable Count (TVC) gives a quantitative idea about the presence of microorganisms such as bacteria, yeast and mold in a sample. To be specific, the count actually represents the number of colony forming units (cfu) per g ( or per ml) of the sample.
A surface viable count is made when the bacterium is best grown in surface culture or on an opaque medium. Ten fold dilutions of the bacterial suspension are made. A suitable volume of each dilution, e.g. 0.1ml, is pipetted on to the surface of each of the plates and at once spread widely with sterile glass spreader or sterile cotton swab. The plates are incubated at 37ºC for 24 hours. The viable count is calculated from the average colony count / plate. A viable count is essential to determine the level of the challenge inoculum.
Only plates (or replicate plates from the same dilution) with 30-300 colonies are counted. Plates with fewer than 30 colonies give statistically unreliable results, while plates with more than 300 colonies are too crowded to allow all the bacteria to form distinct colonies. Usually, more than one dilution in a series is plated, just to be sure that results in a countable range will be obtained. Ignore dilutions giving results outside of the countable range.
The concentration of bacteria in the original sample is calculated as:
CFU ml-1 (or g-1) =
colonies on plate
final plate dilution
Frequently, volumes other than one ml are used to inoculate the plate. For example, 0.1 ml is often used when surface-plating, as larger volumes may not be absorbed by the agar.
For this reason, the size of the inoculum is usually incorporated with the dilution factor to give the "final plate dilution" (d x i). When 1.0 ml of a 10-4 dilution is plated, the final plate dilution is 10-4. When 0.1 ml of the same dilution is plated, the final plate dilution is 10-4 X 10-1 = 10-5.
The formula becomes:
CFU ml-1 (or g-1) =
colonies on plate
d x i
Of the many media available, Muller Hinton agar is considered to be the best for routine susceptibility testing of nonfastidious bacteria for the following reasons.
It shows acceptable batch to batch reproducibility
It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.
It gives satisfactory growth of most nonfastidious pathogens
Meat infusion, beef extract, acid hydrolysate of casein in the medium provide with C, H, N and other trace element sources.
Starch in the medium acts as a protective colloid against toxic substances.
Hydrolysis of starch during autoclaving provides with small amounts of dextrose which acts as a source of energy.
Nutrient Agar consists of peptone, beef extract and agar. This relatively simple formulation provides the nutrients necessary for the replication of a large number of microorganisms that are not excessively fastidious.
The beef extract contains water soluble substances including carbohydrates, vitamins, organic nitrogen compounds and salts.
Peptones are the principle sources of organic nitrogen, particularly amino acids and long chained peptides.
Agar is the solidifying agent.
Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms.
Composition: Chlorexdine Gluconate Solution
I.P 1.5%v/v
Cetrimide Solution (strong)
Specified working range: 1:15 dilution.
Diluent: St. Distilled water.
Composition: Chloroxylenole IP 4.8 %, Terpineol BP 9.0
IP 13.1 % v/v (Alcohol)
Specified working range: 1:20 dilution.
Diluent: St. Distilled water.
Composition: Povidone-iodine
IP 5%w/v. (0.5%w/v available iodine).
Purified water IP.
Media: Muller Hinton Agar (200ml)
Nutrient Agar (40ml)
Diluent: Autoclaved distilled water (50 ml)
Sterile Saline (100ml).
Organism:
Glass ware: Sterile assay tubes (15)
(10 ml) Sterile glass pipette (5 pipettes)
Petri plates (15)
Miscellaneous: Auto pipette (vol. 1000ul, 100ul)
Sterile tips (vol. 1000ul, 100ul)
Cotton Swabs.
Instruments: Incubator at 37ºC.
Votex.
Autoclave
Oven
Laminar Air Flow Unit.
HiMedia Laboratories Pvt. Ltd.
Ingredients: gms/litre
Peptic digest of animal tissue 5.00
Beef extract 1.50
Yeast extract 1.50
Sodium Chloride 1.50
Agar 15.00
Direction of preparation:
Suspend 28.00 gms in 1000ml of distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15lbs pressure (121ºC) for 15 minutes.
The mouth of the nutrient agar flask was flamed to sterilize it and sufficient nutrient agar was poured into the sterile plate (approximately 20ml). The plate was then covered with the lid and then placed on a flat surface and slightly rotated it so that its agar was equally spread on the surface on the plate. The agar solidified in a span on 10 minutes.
2. Mueller-Hinton (MH) agar:
HiMedia Laboratories Pvt. Ltd.
Ingredients: gms/litre
Beef infusion from 300.00
Casein acid hydrolysate 17.50
Starch 1.50
Agar 17.00
Direction of preparation:
38.0 gms in 1000ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121ºC) for 15 minutes. Mix well before pouring.
The mouth of the Mullar Hinton flask was flamed in order to sterilize it and poured sufficiently into the plate (approximately 20ml). The plate was then covered with the lid and placed on a flat surface and slightly rotated it so that its agar was equally spread on the surface on the plate. The agar solidified in a span on 10 minutes.
3. Sterile saline:
To prepare saline water 100 ml of distilled water was poured into the flask and gms of sodium chloride was added to the distilled water. It was then autoclaved at 15 lbs pressure (121ºC) for 15 minutes.
4. Inoculum preparation:
Using colonies taken directly from an overnight (20 to 24 hou r) nutrient agar culture plate, a suspension of the test organism ( S. aureus ) was prepared in sterile saline. The suspension was adjusted to a turbidity equivalent to a 0.5 McFarland standard. This suspension contained approximately 1 to 4 x 108 CFU/ml. Within 15 minutes after adjusting the turbidity of the inoculum suspension, it was used for plate inoculation.
To check the growth of Staphylococcus aureus bacteria on the Nutrient Agar plates it is necessary to check the viable count of the bacteria. The 10 fold dilutions were carried out and 10-1, 10-2, 10-3, 10-4and 10-5 dilutions were plated.
Using colonies taken directly from an overnight (20 to 24 hour) nutrient agar culture plate, a suspension of the test organism ( S. aureus ) was prepared in sterile salinein a sterile assay tube. The suspension was adjusted to a turbidity equivalent to a 0.5 McFarland standard.
5 Sterile assay tubes were labeled as follows - 10-1, 10-2, 10-3, 10-4, 10-5
Using a sterile 10 ml glass pipette 4.5 ml of sterile saline was added to each tube.
0.5 ml of S. aureus suspension was transferred to the first assay tube labeled 10-1 using a 100 – 1000 ul autopipette .
The tube was vortexed and 0.5 ml of culture from this tube was transferred to the second tube labeled 10-2
Again this tube was vortexed and 0.5 ml of culture from this tube was transferred to the third tube labeled 10-3
Similarly dilutions were carried out for the remaining tubes.
After all the dilutions were done, 0.1 ml of the culture (using a 20 – 200 ul autopipette) from each tube was spread plated on respective nutrient agar plates using a sterile cotton swab.
All the plates were incubated at 37ºC for 24 hours.
Microbial Challenge test, which is established by internation practice, is a most widely used to method to evaluate the efficacy of antiseptics. The test is performed over a period of time to simulate clinical scenarios in the process of manufacturing and handling. In this test, samples are inoculated with the test microorganisms and then inspected visually and determine if the antiseptic system is effective. The microbial challenge test method is the classical means to evaluate antiseptic efficacy because it closely mimicks the practical situation
Varying dilutions of the antiseptic were made in sterile assay tubes.
To each dilution add 0.1ml of the culture ( using 20 – 200 ul autopipette ) was added from the inoculum prepared for viable count.
Each tube was vortexed and allowed to stand for 1 minute.
After 1 minute, 0.1ml ( using 20- 200 ul autopipette) from each tube was taken and spread plated on the respective Muller Hinton agar plates using sterile cotton swabs.
All the plates were incubated at 37ºC for 24 hours
1. Viable count:
Dilution
No. Of colonies
10-1
Mat growth
10-2
Mat growth
10-3
Too many to count (TMTC )
10-4
101 X 4= 404
10-5
12
2. Microbial challenge test:
Sample
Dilution
No. of Colonies
A
1:5
0
1:16
1
1:60
0
B
1:10
0
1:20
1
1:50
0
C
1:5
0
1:15
0
Full strength
0
10-4 = 101 x 4 = 404
= 404 x 104 x 10 = 4.04 x 107
~ 4 x 107 cfu/ ml.
10-5 = 12 x 105 x 10
= 12 x 106
= 1.2 x 107
~ 1 x 107 cfu/ ml.
Average: 4 cfu/ ml.
From the viable count the cell density of the initial inoculum was 4 cfu/ml.
Microbial challenge test:
For sample A
In the 1: 15 dilution and the 1: 60 dilution there is no colony growth. Whereas in the 1:16 dilution there is presence of 1 colony of Staphylococcus aureus Bacteria.
For sample B:
In the 1: 10 and 1:50 dilutions there is no colony growth. However in the 1: 20 dilution there is growth of a single colony of the bacteria.
For sample C:
In the dilutions of 1: 5, 1: 15 and full strength dilutions do not have any bacterial growth and therefore no colonies are observed on the plate.
From the observations it can be concluded that the sample A of the antiseptic is effective on staphylococcus bacteria within and beyond its range.
The conclusions that can be drawn from the observations are that the sample B of the antiseptic agent is effective within and beyond its range.
The sample C is effective on the staphylococcus bacteria this conclusion can be drawn from the observation of no bacterial growth on the dilutions of sample C.
Therefore from the results obtained it can be concluded that the staphylococcus bacteria is killed with the help of every antiseptic with varying concentrations in the specific constant time. This proves the effectiveness of the antiseptic. However it is recommended in hospitals and laboratories to change the antiseptic monthly because the bacteria become immune to the antiseptic after a certain period. Therefore with the help of this experiment I have checked the effectiveness of the antiseptics on the Staphylococcus aureus bacteria.
From the Microbial Challenge Test conducted it is observed that all the three antiseptics are effective with and beyond their respective range. However comparing the antiseptics among each other, it can be concluded that the sample C is most effective, since the Sample A and Sample B have 1 colony growth in one of the dilutions. However Sample C does not have any colony growth. The third sample is most effective because its composition includes Povidone- iodine which is not included in the other two samples. However a further question for research has been probed in this experiment to identify if these antiseptics cause any harm to the human body. There is a suspect that these antiseptics are toxic to the human body. Therefore these antiseptics are on account of further research.
The Mechanism of Action and Efficacy Evaluation Methods for Preservatives
Hongyu Xue, Guiqi Yin, Evelyn G. Su
Nanjing Zhongshi Chemical Co., Ltd.
http://www.merck.com/mmhe/sec17/ch188/ch188d.html
http://agrc.ucsf.edu/supplements/immune/immune_system_06.html
http://www.bd.com
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