There are many aspects that must be reviewed when entering the laboratory and there are many regulations that need to be followed to ensure not just your own safety but the safety of your workers around you. Inhalation is one issue that could occur within the laboratory. Within the laboratory many procedures involve the breaking of fluids containing organisms and the scattering of tiny droplets names aerosols. These droplets have the potential to fall contaminating hands and benches while others are very small and dry out immediately. The organisms containing within the aerosol is names droplet nuclei and is airborne and move about in small air currents. If inhaled there could be potential risk of infection so it is important that nothing is inhaled within the laboratories.
Ingestion of organisms is another problem within the laboratory. There are many ways in which organisms may be introduced into to the mouth such as thorough using the mouth to the pipette by direct ingestion, fingers contaminated by handling spilled cultured or from aerosols can potentially transfer micro-organisms to the mouth directly or indirectly by eating, nail biting, licking labels etc. injection is another problem within the laboratory through infectious materials that may be injected by broken culture containers, glass Pasteur pipettes or other broken glass or sharp objects. Through the skin and eye small abrasions or cuts on the skin may not be visible to the naked eye and may allow microbes to enter the body, or splashed of bacterial culture into the eye could result in infection.
This laboratory consisted of working with Hazard group 2 organisms within a containment level 2 laboratory. The hazard level is the level given to the organism which indicates how dangerous the organism could be. Hazard level 2 organisms can cause human disease and may be a hazard to employees although it is unlikely to spread to the community and there is usually effective prophylaxis or treatment available, examples include examples include Salmonella typhimurium, Clostridium tetani and Escherichia Coli.
Within containment level 2 laboratories there are many health and safety procedures to follow, below are examples of health and safety procedures set for containment level 2 laboratory:
• Protective eye equipment is necessary within the laboratory apart from when using microscopes
• There must be specified disinfection procedures in place
• Bench surfaces must be impervious to water, easy to clean and resistant to acids, alkalis, solvents and disinfectants.
• Laboratory procedures that give rise to infectious aerosols must be conducted in a microbiological safety cabinet, isolator or be otherwise suitably contained.
• When contamination is suspected, hands should immediately be decontaminated after handling infective materials and before leaving the laboratory.
• Laboratory coats which should be side or back fastening should be worn and removed when leaving the laboratory.
Within this laboratory glitter bug was applied to the hands and analysed under the light box. Glitter bug is a hand lotion that has a UV fluorescent glow. When place under UV light the glitter bug glows on the places where germs are located which cannot be seen to the human eye.
Loffler’s Methylene blue is a simple stain that was used to stain Saccharomyces cerevisiae.
This is a simple stain which is used for the analysis and understanding of bacterial morphology. It is a cationic dye which stains the cell blue in colour and can be used for the staining of gram-negative bacteria.
Results
Below are the results gathered from the glitter bug before washing our hands. The blue areas indicate where the glitter bug was most fluorescent under the light.
Introduction
Gram Stain
In microbiology one of the most common stains to carry out is the gram stain to understand and observe the differentiation between microbiological organisms. It is a differential stain which can differentiate between gram positive bacteria and gram negative bacteria. The gram-positive bacteria will stain purple/blue in colour and gram negative bacteria will stain red/pink in colour. The results indicating this differentiation can be seen within the variation of the arrangement, cell wall and cell shape structure.
The gram stain has many advantages such as it is very straightforward to partake in, it is cost effective and is one of the quickest methods used to determine and classify bacteria.
The gram stain is used to provide essential information regarding the type of organisms present directly from growth on culture plates or from clinical specimens. The stain is also used within the screening of sputum specimens to investigate acceptability for bacterial culture and could reveal the causative organisms within bacterial pneumonia. Alternatively, the gram stain can be used for the identification of the existence of microorganisms in sterile bodily fluids such as synovial fluid, cerebrospinal fluid and pleural fluid.
Spore stain
An endospore stain is also a differential stain which is used in visualizing bacterial endospores. The “production of endospores is an essential characteristic for some bacteria enabling them to become resistant within many detrimental environments such as extreme heat, radiation and chemical exposure. Spores contain storage materials and possess a relatively thick wall. Possession of a thick wall cannot be penetrated by normal stains either heat must be administered to allow the stains to penetrate the spore or the stain must be left for a longer period to allow penetration. The identification of endospores is very important within clinical microbiology within the understanding and analysis of a “patient’s body fluid of tissue as there are very few spore forming areas. There are two extensive pathogenic spore forming groups which are bacillus and clostridium, together resulting in a variety of different lethal disease such as tetanus, anthrax and botulism.”
The Bacillus species, Geobacillus species and Clostridium species all form endospores which develop within “the vegetative cell. These spores are immune to drying and have the purpose to survive. They develop in unfavourable conditions and are metabolically dormant and inactive until the conditions are favourable for the process of germination returning to their vegetative state.”
The Schaeffer Fulton method is a technique in which is “designed to isolate endospores through the process of staining. The Malachite green stain is soluble within the presence of water and has a small affinity for cellular material potentially resulting in the vegetative cells decolourising with water. Safranin is then applied to counterstain any of the cells which may have been decolourised. Resulting in the vegetative cells being pink in colour and the endospores being green.”
1. The bacteria that were used in this laboratory was Salmonella poona and Bacillus cereus. Both bacteria were identified as gram negative and are rod shaped cells. Other bacteria with identical shape characteristics as Salmonella poona and Bacillus cereus is Klebsiella pneumoniae which belongs to the Genus Klebsiella and the species K. pneumoniae. Another bacterium that has the same shape to those used in thos laboratory is Acinetobacter baumannii which belongs to the genus Acinetobacter and the species Acinetobacter.
2. The loop is sterilised within the Bunsen burner flame by placing the circular portion of the loop into the cold (blue) part of the flame and moving it up into the hot orange part of the flame until it is cherry red. If the loop is placed into the hot part of the flame first the material on the loop (including bacteria) might spurt out as an aerosol and some bacteria may not be destroyed. Once the loop is cherry red this indicates it is sterilised by incineration through dry heat and is then ready for immediate use. If the loop is then laid down or touched against anything it will need to be desterilised again however loops should never be laid on benches.
3. There are many possible problems that could affect a slide smear, for example excessive heat during fixation can result in altering the cell morphology making the cells much easier to decolourise. Another problem could be having a low concentration of crystal violet; this could result in stain cells which are easily decolourise. A third possible problem affecting the slide smear could be excessive washing between the steps as the crystal violet has the ability to wash out with the addition of water when exposed for too long. The last possibility which could affect a slide smear results is excessive counterstaining as it is a basic dye it is possible to replace the crystal violet-iodine complex within gram positive cells with an over exposure to the counter stain.
4. Hand hygiene is a necessity within the laboratories. It is the first line of defence and is considered the most crucial procedure from preventing the spread of hospital acquired infection.
The following steps is the appropriate hand washing technique:
• Wet hands with warm running water
• Enough soap must be applied to cover all surfaces
• Thoroughly wash all parts of the hands and fingers up to the wrist, rubbings hands together for at least 15 seconds
• Hands should then be rinsed under running water and dried thoroughly with paper towels
• Paper towels should be used to turn off taps before discarding the towels in the waste bin.
1. An example of a gram positive bacteria is Propionibacterium propionicus which belongs to the genus Propionibacterium and the species P.propionicus.
An example of gram negative bacteria is Yersinia enterocolitica which belongs to the genus Yersinia and the species Y. enterocolitica.
2. The gram stain has the ability to differentiate between gram positive and gram negative bacteria. Gram positive bacteria possess a thick layer of peptidoglycan within their cell walls but the lipid content of the cell wall is low resulting in small pores which are closed because the cell wall proteins are dehydrated from the alcohol resulting in the CV-I complex being retained within the cells which remain blue/purple. However Gram negative bacteria possess a thinner peptidoglycan wall and a high volume of lipid within their cell walls resulting in large pores that remain open when acetone-alcohol is added. “The CV-I complex is then lost through these large pores. The gram-negative bacteria then appear colourless. Once the counterstain is applied to the bacteria the cells turn pink. This is due to the counterstain entering the cells through the large pores in the wall.”
3. There are many problems which could arise during the production of a bacterial smear. These include having a dirty slide which is greasy or perhaps coated with dirt and dust. Having this will result in unreliable results due to the smear containing the desired microbes washing off the slide during the staining process or when the bacterial suspension is placed on the microscope slide it will not spread out evenly. Another possible problem could be having a smear that is too thick which results in too many cells being on the slide and the penetration of the microscope light through the smear is poor. However, if the smear is too thin then seeking for the bacteria cells is time-consuming.
Germination is also a complex process and is normally triggered by the presence of nutrients (although high temperatures are also sometimes required to break the dormancy of the spore). The events during germination include:
♣ Swelling of the spore
♣ Rupture or absorption of spore coat(s)
♣ Loss of resistance to environmental stresses
♣ Release of the spore components
♣ Return to metabolically active state
Outgrowth of the spore occurs when the protoplast emerges from the remains of the spore coats and develops into a vegetative bacterial cell.
Introduction
The human body and the environment both consist of a vast number and variety of bacteria that are within mixed populations such as within the gut and soil. The bacteria being mixed with such a different variety of populations must be separated in pure culture to investigate and diagnose the identify of each bacterium. The aim of pure culture for bacteria requires that the number or organisms present is decreased until single, isolated colonies are obtained. This can be accomplished through the process of successful streak plate technique or through liquid culture dilutions on a spread plate.
The streak plate technique is used to analyse the purity of cultures that must be managed over long lengths of time. Contamination “by other microbes can be seen through the process of regularly sampling and streaking. The streak plate technique is used in several different aspects such as expert practitioner to begin a new maintained culture through selecting an appropriate isolated colony of an identifiable species with a sterile loop and then going on to grow those cells in a nutrient” broth.
When bacteria in a mixed population are streaked onto a general-purpose medium for example nutrient agar this results in the production of single, isolated colonies however the morphology of the colony does not indicate immediate, reliable means of identification. In practice, microbiologists use differential and selective media in the early stages of separation and provisional identification of bacteria before sub culturing the organisms to a fitting general purpose medium. The identity of the sub-cultured organisms can then be approved using a range of suitable tests.
Selective and differential media are used for the isolation of identification of particular organisms. A variety of selective and differential media are used within medical diagnostics, water pollution laboratorie and food and dairy laboratories.
Differential media normally contain a substrate that can be broken down (metabolised) by bacterial enzymes. The effects of the enzyme can then be observed visually in the medium. Differential media may possibly contain a carbohydrate for example glucose or lactose as the substrate.
Selective media are media that consist of one or more antimicrobial chemicals these could be salts, dyes or antibiotics. The anti-microbial chemicals can select out the specific bacteria while inhibiting the growth and development of other unwanted organisms.
Cysteine lactose electrolyte deficient agar (CLED) is a differential culture medium which is used in the isolation of gut and urinary pathogens including Salmonella, Escherichia coli and Proteus species. CLED Agar sustains the growth and development of a variety of different contaminants such as diphtheroids, lactobacilli, and micrococci.
CLED can be used to differentiate between naturally occurring gut organisms e.g. E.coli and gut pathogens e.g. Salmonella poona in a sample of faeces. There are many advantages of using CLED agar for urine culture, one being that CLED agar is a good discrimination of gram negative bacteria through the process of lactose fermentation and on the appearance of the colonies. Another advantage of using CLED Agar is it is very cost effective and also it inhibits the gathering of Proteus spp which is frequently involved in urinary tract infections.
CLED also possesses lactose as a substrate and a dye names Bromothymol Blue which demonstrates changes in pH. The pH of CLED plates neutral resulting in the plates being pale green in colour. Bacteria such as E. coli that produce the enzyme β galactosidase break down lactose by fermentation to produce a mixture of lactic and formic acid for the pH to become acidic. The colonies and medium then transform into a yellow colour which indicates lactose positive. Lactose negative bacteria cannot ferment lactose due to not possessing the ability to produce β galactosidase resulting in pale colonies on CLED.
MacConkey Agar (MAC) is a selective medium due to the presence of bile salts and crystal violet which inhibits most gram-positive cocci. The bile salts and crystal violet encourage the growth and development of gram positive organism with lactose providing a source of fermentable carbohydrate. MacConkey is designed to isolate and differentiate enterics based on their ability to ferment lactose. Bile salts and crystal violet inhibit the growth of Gram positive organisms. Lactose provides a source of fermentable carbohydrate, allowing for differentiation. Neutral red is a pH indicator that turns red at a pH below 6.8 and is colourless at any pH greater than 6.8.
Organisms that ferment lactose and thereby produce an acidic environment will appear pink because of the neutral red turning red. Bile salts may also precipitate out of the media surrounding the growth of fermenters because of the change in pH. Non-fermenters will produce normally-colored or colourless colonies. In MacConkey agar, the substrate is lactose which is fermented by lactose positive bacteria e.g. E. coli to lactic acid and formic acid resulting in the medium being acidic. The dye neutral red then changes colour and colonies of E. coli are now violet red. Lactose negative bacteria are describes as possessing pale colonies and therefore mac can be used to select out and differentiate between naturally occurring gut organisms and gut pathogens.
1. Figure 13 elicits how the majority of the colonies used in laboratory 3 took up the entire colony edge and where flat in the elevation of the colonies. The streak plate method can obtain single colonies through firstly streaking the portion of the agar plate with an inoculum and then streaking successive areas of the plate to dilute the original inoculum so that single colony forming units (CFUs) will give ruse to isolated colonies.
2. Potential problems that could lead to the production of unsuccessful plates or slants could be that when sterilising the loop, it was placed in the inner blue flame and not given time to cool down being instantly placed directly into the plate, killing all bacteria within the plate. Another problem could be insufficient flaming between the quadrants leading to the loop not being sterile leading to contamination of organisms.
3. A Bacterial cell is a microscopic single- celled organism which thrive in diverse environments. A Bacterial colony is a discrete accumulation of a significantly large number of bacteria, usually occurring as a clone of a single organism or of a small number.
4. Refer to Figure 15 and 16
5. Cled it is a solid medium used in the isolation of gut and urinary pathogens including Salmonella, Escherichia coli and Proteus species. CLED contains lactose as a substrate and a dye called Bromothymol blue which indicates the changes in pH. Prior to inoculation, plates of cled are pale green in colour. This is due to the pH of the plates being neutral. Bacteria such as E. coli that produce the enzyme β Galactosidase break down lactose through the process of fermentation to produce a mixture of lactic and Formic acid so that the pH is acidic. Resulting in colonies and medium turning yellow (lactose positive). Lactose negative bacteria e.g. Salmonella poona is unable to ferment lactose due to them not being able to produce β Galactosidase and usually producing pale colonies on cled. Cled can therefore be used to differentiate between naturally-occurring gut organisms.
6. From the results, we can conclude that the bacterium that fermented lactose was Escherichia. Coli and the none fermenting bacterium was Salmonella poona.
7. Mannitol Salt Agar (MSA) is utilised as a selective and differential medium in the process of isolating and identifying Staphylococcus aureus from clinical and non clinical specimens. Mannitol Salt Agar contains the carbohydrate mannitol, 7.5% sodium chloride and the pH indicator phenol red. Phenol red is yellow below p.H 6.8, red at pH 7.4 to 8.4 and pink at 8.4. the sodium chloride makes this medium selective for staphylococci since due to most bacteria not being able to live in such levels of salinity.
The pathogenic species of staphylococcus ferment mannitol and thus produce acid. This acid then turns the pH indicator to a yellow colour. Non-pathogenic staphylococcal species grow however there is no colour changed produced.
The formation of yellow halos surrounding the bacterial growth is the predicted evidence that the organism is a pathogenic Staphylococcus. Significant growth that produces no colour change is the presumed evidence for none pathogenic Staphylococcus. Those staphylococci that do not ferment mannitol produce a purple or red halo around the colonies.
A viable count
A viable count is a method for estimating the number of bacteria cells in a specific volume of concentration. The method relies on the bacteria growing a colony on a nutrient medium. The colonies then become visible to the naked eye and can then be counted. For accurate results the total number of colonies must be between 3-300. Fewer than 30 indicate the results are not stastically valid and are unreliable. more than 300 colonies often indicate an overlap in colonies and imprecision in the count. To establish that there is an appropriate final figure for the total colony count several dilations are normally cultured. The viable count method is used by microbiologists when undergoing examination of bacterial contamination of food and water to ensure that they are suitable for human consumption.
Serial Dilution
A serial dilution is the process of consecutive dilutions which are used to reduce a dense culture of cells to a more applicable concentration. within each dilution the concentration of bacteria is reduced by a certain amount. through calculating the total dilution over the entire series the number of initial bacteria can be calculated. After the dilution of the sample an estimation of the number of bacteria visible is carried out using the surface plate count known as the spread plate technique and the pour plate technique. Once incubated the colonies are then counted and an average is calculated. The number of viable bacteria per ml or per gram of the original sample is also calculated however this is calculated on the principle that one visable colony is the direct result of the growth of one single organism. Nonetheless bacteria has the capability of clumpimg together and this could result in a colony being produced from a clump. For that reason counts are expressed as colony forming units (cfu) per ml or per gram as this gives the explanantion as to why counts are estimations.
Spread plate technique
The spread plate technique is used for viable plate counts for when the total number of colony forming units on a single plate is counted. There are many reasons as to why the spread plate technique is so useful within microbiology for example is can be used to calculate the concentration of cells in a tube from which the sample was initially plated. The spread plate technique is also routinely used in enrichment, selection and screening of experiments. However, there are some disadvantages for when using this technique such as crowding of the bacterial colonies could make the enumeration much more challenging.
Pour plate technique
The pour plate method is used in the counting of the coloy-forming bacteira present in a liquid form. The pour plate has many advantages fo example it allows the growth and quanitifation of microaerophiles as there is little oxygen within the surface of the agar, and identification of anaerobes, aerobe or facultive aerobes is much easier as they have the ability to frow within the media. Howver there are a feew disadvatges in using the pour plate technique for example the temperature of the medium needs ti be tightly regulated. If the temperature is too warm the mirocrogansims will die and if the temperature is too cold the agar will clump together which can sometimes be mistaken for colonies.
Introduction
In microbiology understanding the characteristics that bacteria possess is critical to the knowledge and understanding of microbiology. To enable a full understanding of the characteristics bacteria possess they undergo simple tests named primary tests which can be used to establish if the cells are gram negative or gram negative cells, if the cells are rods or cocci shape and if the bacteria is catalase positive or catalase negative.
The catalase test is a primary test which is used in the detection of catalase enzymes through the decomposition of hydrogen peroxide resulting in the release of oxygen and water as demonstrated by the equation below:
2 H2O2→2 H2O + O2
Hydrogen peroxide is produced through various bacteria as an oxidative product of the aerobic breakdown of sugars. However, it is highly toxic to bacteria and could lead to cell death. The catalase test serves many purposes such as differentiating between the morphologically similar Enterococcusor Streptococcus which is catalase negative and Staphylococcus which is catalase positive. The test is also valuable within differentiating between an aerobic and obligate anaerobic bacterium and can be used as an aid within the identification of Enterobacteriaceae.
The oxidase test is also an example of a biochemical primary test which is used in the identification of if bacteria produce cytochrome c-oxidase which is an enzyme of the bacterial electron transport chain.
Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase which both catalyse the transport of electrons from donor compounds such as NADH to electron acceptors which is usually oxygen. If present, the cytochrome c oxidizes the reagent (tetramethyl-p-phenylenediamine) to (indophenols) producing a purple color as the end product. When the enzyme is not present, the reagent remains reduced and is colourless.
Organisms which are known as oxidase positive are- Pseudomonas, Vibrio, Brucella, Pasturella, and Kingella. Organisms which are oxidase negative are Acinetobacter, Staphylococci, Streptococci and all Enterobacteriaceae.
Primary tests are helpful in the understanding of the initial characteristics bacteria possess. However more advanced methods may be used to finalise the identification to the level of Genus and Species to enable treatment for patients and to enable appropriate action to be taken to prevent any further transmission of infection. Laboratories today now rely on rapid id kits which analyse the biochemical aspects of bacteria and this is known as bio typing.
Rapid identification kits are used for the identification and differentiation of different bacteria. The ID32E is commonly used in the identification of members of the Enterobacteriaceae. There are two types of kits one is IDSTAPH which us used in the identification of members of the staphlycococcaceae while the IDSTREP strip is used in the identification of streptooccaceae. The kits consist of wells which contain dried substrates such as sugars or amino acids. These dried substrates are then reconstituted through the addition of saline suspension of bacteria. The results are then read on a computer profile which is linked to an identification software. From then the genera and species can be analysed and differentiated from each other.
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