MLT 1 Lab MLT 1 Lab

MLT1 Lab

MLT1 Lab

Task1

Inthe laboratories, there are several techniques used in mounting ofslides. These techniques include, fresh wet mount techniques, directstaining and indirect staining methods. Using a fresh wet mountslide, bacterial shapes are able to be determined, however, they arenot distinct and their borders are vague (Cromack et al., 1979).Liquid is normally formed and it resembles the color of the cells.Direct staining has precise and distinctive images that are purple incolor. The indirect staining method utilized two dyes, when usingCongo Red stain, the epithelial cells are stained rather than itsbackground. S. cerevisiae cells are easily seen while the dentalplaque smear is not visible at all. When staining indirectly usingcrystal violet, all the images of the three cells are shown indetails when viewed on a virtual microscope.

Directstaining is a type of staining that stains the bacterial cells. Itemploys the use of basic byes like crystal violet, carbol fuchsin andmethylene blue. These dyes have positive ions that normally reactwith negative ions on the microbes, because of the opposite charges,the microbe will be stained directly. Indirect staining stains thebackground of microorganisms. The dyes used in staining includesodium eosinate and negrosin. These dyes are acidic thus they possessa negative charge. Therefore the bacteria will repel the dye’scharge rendering it colorless, instead, only its background will becolored (Cromack et al., 1979). This method provides a more detailedand precise image of the bacterial cells.

Bothyeast and plaque cells are similar in that they are round in shape.Yeast cells are nucleated, round, flat and smooth. Their cells areclosely spaced making them to have a sausage like appearance. On theother hand, cells in the plaque are spaced widely. Plaque smear has amultiple bacteria species in it (Cromack et al., 1979).

Dentalplaque bacteria vary in shape and sizes. The bacteria affecting themouth are majorly from the streptococcus species. They are gramnegative and their population has bacteria that are spindle shaped,cocci and filamentous rods. The bacterial cells in the cells areisolated and their round shapes helps them in adhesion to the teethand gum.

Task2

Aerobesare organisms that grow in an oxygen media. Aerobes are classifiedinto two categories, facultative aerobes and obligate aerobes.Facultative aerobes are organisms that are able to utilize oxygen forthe provision of ATP. In hostile conditions, they shift tofermentation. Obligate aerobes are dependent on oxygen supply forgrowth. These microorganisms metabolize with presence of oxygen.Because they base their concentration mostly on aerobic respiration,they manufacture ATP that yields energy of higher level than that offermentation.

Temperatureaffects microorganisms in the laboratory either chemically,nutritionally or in growth. In a laboratory, there are differentmicroorganisms with different range of temperature requirements. Theyare grouped into three categories, this include, thermophilic,mesophilic and psychrophilic. Thermophilic microbes require hightemperatures for metabolic reactions. They grow at a temperatureabove 45 degrees. Mesophilic microbes grow at medium temperatures.Their ideal temperature for is 25 – 40 degrees. Psychrophilicmicroorganisms grow at low temperatures, that is, temperatures thatare below 20 degrees Celsius. Microorganisms used in this laboratory(L. acidophilus, E. coli, S. cerevisiae, S. epidermidis) aremesophiles, this is because the live in the body which has a moderatetemperature. These organisms are the type that either stay in thesoil or in human beings gut flora, urinary tract and the intestines.

Microbialgrowth control involves inhibiting their progress. Controllingmicrobial growth is necessary for elimination of possible causes ofinfection from the microorganisms. In the laboratory, controllinggrowth is essential in experiments so as to get useful results.Growth control of microorganisms involves either killing them orinhibiting their growth. Control of growth involves the use of cidalor static agents. The cidal agents kills the microorganisms while thestatic agents inhibit their growth. Methods used are sterilizationand heating. Sterilization kills all the bacteria in a surface, thisrenders the surface sterile and safe to be handled.

Thereare basic forms of culture media. They are classified accordingly totheir nutrient requirements, consistency and functions. In theconsistency media, they are grouped into solid, liquid, biphasic andsemi-solid media. The consistency media is used to grow a wide rangeof bacteria and is good in giving large numbers of bacteria. Infunctional media, they are grouped in accordance to the function theywill perform, the can either differentiate bacteria from pathogens byaddition of dyes, food substrates or antibiotics. Nutritional mediaeither grows fastidious or non-fastidious bacteria. Fastidiousbacteria needs complex nutrients like blood agar for growth.

Therewas no growth on the tube after 24 hours, this is because theincubation temperatures were maximum. The microbes that were in useare mesophilic and require moderate heat. After 48 hours, growth wasobserved on the sides of the tube, this is because microbes from theair entered the tube. These microbes tolerate high temperatures.

Task3

Mediaare used for the growth of microorganisms. Enriched media is rich innutrients than can support growth of a wide range of bacteria. Thesebacteria include both the fastidious and the non-fastidious. Definedmedia are media whose nutrient concentration is measured. It ismostly used in experiments for research purposes. Complex media isused to grow fastidious bacteria, it is rich in sugar, organicnutrients and minerals (Cromack et al., 1979).

Selectivemedia supports the growth of only one type of microorganism. Toachieve this, the growth of other microorganisms is inhibited. Thisis accomplished by addition of substrates, dyes and antibiotics. Thesubstrate used should be specific to the type of microbe needed.Differential media distinguishes related groups of microorganismsfrom one another. These organisms will show certain growth patternsand characteristics when chemicals and dyes are added to the media.

Asolid media is mostly reliable for one to obtain a pure culture. Themethod that will be involved when placing a media in a solid agar isthe streaking method. This method will disperses the individual cellsin the solid. This will facilitate isolation leading to the formationof discrete colonies by each individual cells.

Microbialgrowth is in large numbers in the streak plate method than the pourplate method. Thestreaking plate method is more qualitative andhomogenous. The density of the streaks throughout the streaks will beequal. The pour plate method will have decreasing number of coloniesand some of the colonies will be observed stuck on the middle.

S.epidermis appears as diploccoci or cocci in shape. Their cocci arearranged in that they form clusters that resembles grapes. Theclusters are cohesive colonies whose diameter is about 1 – 2 mm.the color they formed after an overnight incubation was white. Inother occasion, it forms flat colonies.

Task4

Gramstaining is used to distinguish the gram positive and the gramnegative cell walls. A Gram positive cell wall has a thickpeptidoglycan layer that retains the purple color of crystal violetwhen stained. Gram negative cell wall has a thin peptidoglycan layerthat stains pink when counterstained with safranin.

Inthe gram staining procedure, iodine is normally used in the secondstep. It acts as a mordant whose purpose is to make the stain adheretightly to the stained cell. A mordant is a constituent that works asa dye binder, in chemistry it is useful for strengthening of stainson tissue preparations. Acetone alcohol is used on the third step itacts as a decolorizing agent. The acetone alcohol will wash away thecrystal violet stain in the gram negative microbes so that it can bestained with safranin.

E.Coli is a gram negative microorganism this microbe when stained withcrystal violet, its thin peptidoglycan layer does not permanentlyretain the purple color. Acetyl alcohol washes the purple color away.Addition of safranin after the decolorisation process will make itpink. S.cerevisiae is a fungi that is grouped under the gram negativemicrobes, it contains ascospore which stains pink with the gramstains. L. acidophilus and S. epidermidis are gram positive, theyhave a thick peptidoglycan layer that when stained with crystalviolet, they permanently retain the purple color. The purple staincannot be washed away during the decolorisation process. Thesemicrobes have severe effects in the infections the cause. The mostcommon infection associated with E.coli is the Urinary TractInfecions. The most vulnerable persons are the aged and those thatuse urinary catheters. S. epidermidis is the most common primaryinfection associated with hospital equipment, they attach tocatheters, implant devices, and artificial hearts. L. acidophilus iscommonly associated with bloating of the stomach. These bacteriabreak down sugars in our guts and they produce gas like methane andcarbon dioxide. Accumulation of these gases results to bloating ofthe stomach. S. cerevisiae is a type of yeast that is mostlyassociated with fungal infections, the infections results to sepsis,pneumonia and liver abscess.

Task5

Methylred is a test that is used to determine if a microbe can achievemixed acid anaerobic respiration with presence of glucose. Thequantity and type of products synthesized by the fermentation is theone key factor that is used in distinguishing microbes. Methyl redacts as an indicator of acidity or alkalinity.

Voges-Proskaueris used to determine the ability of a microorganism to yield 2,3-butanediol as a result of glucose fermentation. The reagents usedin this experiment include, potassium hydroxide solution andalcoholic a-naphtol. These two are mixed to enable them determine thepresence or formation of 2-3, butanediol.

MethylRed/ Voges- Proskauer is used to determine the route which a glucosemolecule will be used in a microbe. When E.coli and S.epidermidiswere used in this experiment, E.coli showed a positive result inmethyl red test, this is because it lacks the ability to fermentglucose. E. coli will test negative in the Voges-Proskauer test, thisis so because it produces acid that cannot yield 2,3-butanediol. Sepidermidis tests positive on both the methyl red and Voges-Proskauertest .S. epidermidis yields 2,3-butanediol as a results offermentation. Both S. epidermidis and E .coli produces quantifiableamounts of acidic byproducts. E. coli produces succinic acids.

Glycolysisis a metabolic route that is used in metabolism of glucose by severalmicrobes. Organisms have different pathways for glucose metabolismdue to the variations in their environments and the energy they willrequire. An organism in an oxygen deprived environment will depend onfermentation to synthesis energy.

Task6

Flagellaare distinguished accordingly into four groups according to theirarrangements. These arrangements include monotrichous,lophotrichous, amphitrichous ant peritrichous. Monotrichous microbeshave a flagellum which extends from one pole of its cell.Amphitrichous microbes have one flagellum extending from both sidesof its cell. Lophotrichous flagella arrangement is whereby severalflagella protrude from either one or both sides of the cell.Peritrichous microbes have multiple flagella all over their cell.

Aninoculating needle is the appropriate laboratory to determinemotility of microbes while a transfer loop is used in the picking ofspecimens. When a needle is used, it forms a distinct stab line thatwill facilitate the growth of microbes along it. The microbes areknown to be motile if there is growth along the needle.

E.coli observations showed a positive motility. They moved in clustersalong the needle on the motility tube. E .coli have peritrichousflagella, this facilitates their motility especially when they haveinfected someone. E. coli are found on the urinary and intestinaltracts. On the other hand, S.epidermidis are non-motile, for theirmobility, they adhere to plastic equipment like catheters where theywill infect their host. When the two microbes are placed in a wetmount preparation, and then observed, E.coli showed motility.S.epidermis did not move.

Task7

Fermentationprofile.

Phenol Red Test

microorganisms

Fructose

Glucose

Mannitol

S. epidermidis

yellow

yellow

yellow

Saccharomyces cerevisiae

yellow

yellow

Dark pink-red

Thefermentation tubes are not to be incubated for more than 24 hoursbecause beyond it, the organisms will have consumed all the sugars.This will make them to change behavior and respire anaerobically andit may give a negative result.

Phenolred acts as an indicator in detecting the fermentation process. Whenfood substrates are consumed, the microbes produces acidic byproducts, this will change the color of the media to yellow. Withoututilization of the carbohydrate, the media is neutral, it results torise in pH, and the resulting media turns to fuschia.

Bacteriautilizes carbohydrates in their metabolism for the production ofenergy (Cromack et al., 1979). Bacteria ferment carbohydratesdifferently because they have diverse enzymes in their metabolism.Some bacteria are able to use complex carbohydrates such mannitol andfructose while others utilize simple sugars like glucose (Cromack etal., 1979).

Task8

Amedium can either be isotonic, hypertonic or hypotonic. Whenmicrobial cells are placed in these different media, they will behavein distinguished ways so as to either gain or expel water from theircells. When a cell is isotonic to a media, the salt or sugarconcentration in it is equal to that of the media. Hypertonic mediahave more concentration of salt than that of the microbial cell,water will move out of the cell to the media through osmosis thiswill lead to shrinking of the cell or plasmolysis. When a media ishypotonic, it has less salt concentration than that of the cell.Therefore, the cell will gain water and swell.

WhenStaphylococcus epidermidis and Saccharomyces cerevisiae were placedin different concentrations of sodium chloride solutions, thefollowing were the results. When S. cerevisiae was placed in 1percent sodium chloride solution, there was moderate growth thatappeared to be cloudy and even. In 7 percent sodium chloridesolution, there was minimal growth that grew on the sides of thetube. The results for S. epidermidis were as follow moderate growthin the 1 percent sodium chloride solution, though, it was not muchclouded. Moderate growth was observed in the 7 percent sodiumchloride solution. No growth in 15 percent sodium chloride solution.

S.cerevisiae and S. epidermidis grows in varying sodium chlorideconcentrations. They grow in 1 percent and 7 percent sodium chloridesolutions because they have a mechanism that enables them tocounteract with the effects of these salts concentration. Theirmechanism takes place with the help of osmotic pressure. In 15percent sodium chloride solution, the microbes will not grow becausethey have not reached levels to tolerate such salt concentrations.

Task9

Selectivetoxicity is the effectiveness of a drug or a chemical, whereby whentaken by an infected host, it will only have an effect or kill themicrobe (Cromack et al., 1979). These chemicals have no harm ortoxicity to the host.

Selectivetoxicity is of great importance to microbial agents because theseantibiotics act in such a way that they inhibit growth or killmicrobes in the body of host. These agents have minimal or no effectto the host in which the microbes exist (Cromack et al., 1979).

Broadspectrum antimicrobials kills a wide range of microorganisms, thisinclude both the gram negative and gram positive microbes. Narrowspectrum antimicrobials have effects on gram negative microbes.

Narrowspectrum and broad spectrum antimicrobials have advantages anddisadvantages. Narrow spectrum antibiotics works on specificity, inthat, it will have an effect on only one type of bacteria in thebody, this will therefore lead to non-collateral damage of the body’sfriendly bacteria (Cromack et al., 1979). Narrow spectrum drugs havea limitation in that one should know the specific bacteria in thebody so as to be treated. Failure to recognize the bacteria in thebody will lead to reinfection. Broad spectrum antimicrobial treats awide range of microbes including the helpful flora. It has anadvantage in that it can treat a wide range of infections.

Microbesdevelop resistant to antibiotics when they are not used accordinglythey are either overdosed or under dosed. When these drugs are usedincorrectly, a population of bacteria will remain in the body, theywill develop a strain for resistance and therefore the use of thesame drug will not be effective. These body microbes have severalmechanisms that facilitate resistance they will tend to change theirpermeability, cell envelope or their efflux and degradation (Cromacket al., 1979).

Whenperforming the Kirby-Bauer test for S. epidermidis, the bacteria isswabbed evenly on a nutrient agar plate. The culture is inoculatedand grown overnight. Three filter paper disk infused with theantibiotics are then placed on the culture plate. Three zones arethen marked due to differences in the antibiotics drugs being used onthe disk. In this case, novobiocin, penicillin and gentamicin areused. The zone containing novobiocin is marked N, penicillin P, andgentamicin G. After the disks are placed further away from eachother, the plate is incubated for 24 hours for better results. Thefollowing were the results no growth around the disks in zone N andG. growth on zone P. when the results were analyzed, it showed thatS. epidermidis shows both sensitivity and resistance to antimicrobialdrugs. The microbe is sensitive to novobiocin and gentamicin drugs.Circles are formed around disk G and N.. This circle is known as zoneof inhibition. This shows that S. epidermidis was not able to growaround the disk so they diffused away from the disks. On the diskimpregnated with penicillin, there was growth around it, thisconfirms the resistance of S. epidermidis to penicillin.

Task10

Transmissionof disease requires three elements a reservoir, a host prone to theinfection and a means of exit. Reservoir is the agents causing theinfections by invading the host. When someone is already infected,the bacteria will have adverse effects on the person and it mayreplicate several times when inside the host. Microorganisms willfinally exit the host.

Apotential host is prone to infection due to several factors, thisinclude the level of immunity, age of the host and the genetic kindof the host. Immunity in individual ranges from those with low tohigh. Immune individuals are able to fight any slight infections.Immune deficiency decreases the body’s ability to fight infections.Infants are highly susceptible to infections than adults (Cromack etal., 1979).

Pathogensare disease causing microorganisms transmitted through three modes.The first mode is the contact transmission. This transmissioninvolves direct contact of infected persons or objects it can eitherbe direct through physical contact with susceptible persons orindirect through contact with inanimate objects. Infectious agentscan also be transmitted through droplets found in the coughs, sputumor through sneezing. These droplets are light and when inhaled, theywill settle on the host nasal membranes. The second transmission isairborne. Infectious agents are transmitted through air and with thehelp of wind they are propelled to prone hosts. The thirdtransmission is the host this will depend on the hosts’ ability tofight infections.

Fomitesare inanimate objects through which pathogens settle until they finda potential food source.8 environmental sites were located to testfor fomite growth. The following were the results when they wereplaced on dishes to grow

  • Doorbell numerous growths that is all over the petri dish. Its colonies are isolated and cream colored.

  • School toilet door knob cream colored circular colonies that are raised and have smooth surface. The growth of these colonies is large with only a few colonies isolated.

  • Refrigerator handle medium growth with 10 secluded colonies. The colonies are raised and cream in color.

  • Oven no growth seen.

  • Toilet base moderate growth, colonies are isolated, small, cream in color, circular with dull surfaces.

  • Toys minimum growth that is cream colored, shiny and raised colonies.

  • Picture frame had moderate growth with large colonies that are cream colored with a dull appearance.

  • Pulling cart keys numerous growths that are cream in color, raised, circular and a smooth surface.

Therewas large amount of growth in some objects because they were exposedto the environment and bacteria can easily attach to them. Theseobjects are found in public places and people infected can attachbacteria on these objects. These objects include school toilet doorknob, doorbell and the pulley cart. There was medium growth on therefrigerator handle, toilet base and picture frames. These places arenormally disinfected and bacteria attaches to them from the air.Minimum growth is observed on the toys and no growth is seen in theoven, this is because of the high temperatures which cannot betolerated by microbes.

Task11

Microbesthrive in environments that have various conditions and temperatureranges. In fact, they are found almost everywhere. Microbes are foundin the soil, water and air. Microbes that are found on the ground maybe in the compost pits, frozen grounds or some exhibit symbioticrelationship with the roots. Inside our body, microbes live in ourdigestive tract, mouth and our skin. Microbes tend to live indifferent temperatures, some high while others in lower temperatureenvironments.

Microbesplay a vital role in our ecosystem. These roles include biosynthesis,biodegradation, waste water treatment, food processing and recyclingof nutrients. Microbes take up nutrients by feeding on carcass. Theydecompose the dead matter to release nutrients that is trapped on thebody. These organisms also fix nitrogen in the nitrogen cycle. In thefood industry, microbes ferment, brew and bake breads. The wastewater has to undergo several stages of treatment for its release inwater bodies like rivers, microbes helps in the final process byremoval of organic waste. In the end, they release methane as abyproduct which is useful in heat generation. Microbes biodegradesnutrients by decomposing organic wastes from our homes andindustries. When the wastes are decomposed, they are used by plantsfor growth.

Microorganismsexhibit several growth characteristics in the media they are placedto grow. When observing growth on air dish, water dish and soil dish,there are distinctive number of colonies, shape and colorcharacteristics. First, when the growth on an air dish is observedafter a specified duration, most of the growths are coccus, theothers are large, cream and coccobacilli in shape. The total numberof colonies equals to six. In the water dish, there are more coloniesthat are large while some are tiny. The colonies are coccus shapedwhich is cream in color while some of the coccus appears as blackshaped dots. The final observations were made in the soil dish thisdish too had several colonies. The colonies though were tiny, coccusshaped and cream in color. From the above observation, the growthshave common similarities. The colony in each and every plate iscream/whitish and they are coccus in shape. The growths have a slightdifference in that they have different numbers of colonies.

Microbesare designed in such ways that enable them to adapt to differentenvironmental conditions or situations. Most of microbes adaptabilityis genetically based. The change can either be reversible or it canthrive to succeed the other generation. Some Microbes have developedresistance to antimicrobial drugs. The gram negative bacteria are allresistance to penicillin this is because they have developed acoating that does not allow the penetration of penicillin. Microbesadapt to environmental conditions like temperature, pH andalkalinity. In a more viscous environment, they form swarmer cellswhich help in their mobility. This adaptation makes them to beattached on fomites’ which further infects humans. Microbes altertheir genome composition this makes them to be unidentifiable byantimicrobial drugs.

Changesin the environmental conditions causes increase in diseasetransmission. Careless disposal of industrial or home wastes makesthe microbes to be widely spread. They are quickly transferred to theair, objects and surfaces at our home. In some situations, thebacteria may lack its host this renders their reintroduction to anew host population.

Task12

Conidiosporesand sporangiospores are produced by molds. They are all asexualspores. Conidiospores are formed in chains at aerial hyphae endings.They have a unicellular spore that lacks a sac. Sporangiospores areformed at the endings of aerial hyphae called sporangiospores. Theirspores are enclosed within a sac known as sporangium. Conidiosporesare produced by aspergillus while Rhizopus stolonifer producessporangiospores.

Azygospore is a reproductive cycle of most fungi. They arethick-walled and are normally formed through fusion of haploid cellsthat results to a diploid form. The zygospore phase is responsiblefor production of sporophytic phase. Rhizopusstolonifer produces zygospores during its sexual reproduction. Itsthick wall enables the mold to withstand harsh environmentalconditions.

Thetwo food substrates had mold growing on them after duration of time.On the bread, several colonies were observed. The colonies were whiteand they resembled a fluffy cotton. Other colonies formed were yellowand green. The fruits shows two colonies that are yellow and green.The inner surface of the fruit is affected but no mold is observed onits cover. The colonies on the fruits were in small circles that hada nobly texture.

Breadand fruit are affected with different types of molds. The molds inthe bread are Aspergillus Niger and Rhizopus stolonifer while themoulds in the fruit are Botrytis Cinerea and Alternaria. When thefood subtrates containing these molds are observed for morphologicalcharacteristics on a wet mount, the hyphae, rhizoids and stolons areseen. When the wet preparation is stained, the spores in the moldsare seen, this is because the stains makes the spores to be coloredhence more visible.

Mostof the fungi normally attach on the mucosal membranes inside bodyorgans and on the skin, when they replicate, they increase in number.They outnumber the neutrophils which fight against them. Thereplication mechanism makes fungi of distinct forms thus theimmunocompromised individual is greatly affected. Most fungireplicate within phagocytes, this helps in their survival. Fungi thatreplicate outside blood cells can be easily cleared out through hostdefense.

References

Cromack,K., Sollins, P., Graustein, W. C., Speidel, K., Todd, A. W., Spycher,G., … &amp Todd, R. L. (1979). Calcium oxalate accumulation andsoil weathering in mats of the hypogeous fungus Hysterangium crassum.Soil Biology and Biochemistry, 11(5), 463-468.