Microbiology Lab Final Observations and Results/ Review Questions

Explain the reason for the use of different medium blanks in adjusting the spectrophotometer prior to obtaining A readings.

Uninoculated media tubes, representative of themedia in which the cultures have been grown, are used as blanks. As the different media exhibit variations in color, the blanks must be used to standardize the spectrophotometer to 100% T prior to obtaining the A readings of the cultures.

A patient exhibits a boil on his neck. You, as a microbiology technician, are asked to identify the causative organism and determine whether it is pathogenic. Describe the procedure that you would follow to make this determination.

A boil is usually the result of a staphylococcal or a streptococcal infection. The exudate should first be cultured in a broth medium, followed by streak-plate inoculations on blood and mannitol salt agar plates for the isolation of discrete colonies. If the etiological agent of the boil is Staphylococcus aureus, a yellow halo will be present surrounding some of the colonies on the mannitol salt agar plate, and beta-hemolysis will be evident on the blood agar plates. If the causative agent is a pathogenic streptococcus, evidence of beta-hemolysis will be present on the blood agar plate; however, none of the colonial growth on the mannitol salt agar plate will exhibit a yellow halo.

While you are working in your garden, a tine of the pitchfork accidentally produces a deep puncture wound in the calf of your leg. Discuss the type of the infectious process you would be primarily concerned about and why?

A deep puncture in calf of leg caused due to pitchfork can get infected with tetanus. C tetani spores can enter the body through a deep puncture or wound in the skin. Deep sites that lack oxygen provide ideal environment for growth of these spores into bacteria.Clostridium tetani bacteria produces tetanus toxins which block nerve signals from spinal cord to muscles leading to acute onset of hypertonia.

What are some of the microorgansisms termed "normal flora," and of what value are they to the well being of the host

Certain microbes are found associated with many human body parts such as the skin, the intestinal tract, the oral cavity, etc. These microbes are found in normal conditions and do not pose a health risk and refferred as the normal "flora". Many of these microbes have symbiotic relationships with the host system where the host provided nutrinets to the microbes and the resident normal flora providing protection from new microbial entry and in some cases, even scrteting important compounds such as vitamins that host requires

Explain the advantages of using A readings rather percent T as a means of estimating microbial growth

Absorbance is directly proportional to the amount of microbial growth, whereas percent Tis inversely proportional to the number of cells present.

Why are proteins and amino acids considered to be natural buffers?

Proteins and amino acids, because of their amphoteric nature, serve as natural buffers, as they can either donate or accept H+.

Explain the mechanism by which buffers prevent radical shifts in pH.

if the pH decreases, the salt of the weak base will accept excess H+, forming a weak acid. If the pH increases, the weak acid will donate H+, thereby forming a weak base.

Explain the effects of temperatures above the maximum and below the minimum growth temperatures on cellular enzymes.

Enzymes will be denatured above the maximumgrowth temperature. Below the minimumgrowth temperature, enzymes are inactivated

If 0.1 ml of a 1 x 10^-6 dilution plate contains 56 colonies, calculate the number of cells per ml original culture

(1 x 10^-6)= 1, 000,000 56 x 1,000,000 / 10= 56,000,000/10= 5x 10^5

A 35-year old female underwent serious abdominal surgery involving extensive bowel resection. She was maintained postoperatively on a regimen of intravenous broad-spectrum antibiotics. Three days postoperative she spiked a fever without a clear source. She complains of vaginal discomfort. Blood cultures reveal the presence of an ovoid cell that reproduced by budding. A) Based on this observation, what do you think this organism is? B) Is it part of the normal flora in humans? C) How did the treatments with broad-spectrum abitbiodies predipsose the patient to infection with this organism?

A) the ovoid cell that reproduced by budding would be a fungus, most probarly candida albicans B) the fungus Candida albicans is a part of the normal flora in humans C) The treatment with broad-spectrum antibiotics predipsoses the patient to infection with Candid albicans because in the nromal conditions, the other normal flora keeps a check on the growth of this fungus. The prolonged broad-spectrum antibiotic treatment resulted in teh loss of normal flora and helped in the fungus to take the oppurtunity to cause the infection

A 6- year- old female is taken to her pediatrician for a checkup. As the doc-tor takes the child's history, her mother reports that the child had a severe sore throat several weeks earlier that regressed without treatment. Upon examination the pediatrician notes that the child has a systolic heart murmur consistent with mitral insufficiency and suspects that she has rheumatic fever. A) How was the earlier pharyngitis related to the subsequent development of reumatic fever? B) rheumatic fever is diagnosed on clinical and serplogical findings. What test should be done to diagnose rheumatic fever? C) How are pateince with rheumatic fever treated?

A) The causeative agent of rheumatic fever is same as the one that causes pharyngitis, which is streptococccal pyognes. However, it takes more time or the rheumatic fever to be induced as a proteins known as the M protein should be produced by the pathogen for the onset of the rheumatic fever. Thus the earlier pharyngaitis is related to the subsequent development of rheumatic fever B) The test that should be done to diagnose rheumatic fever include a physical esxmaination, elctrocardoimgram and serological test to detect ciruclating anti-streptococcal bacterial anitbodies C) Rheumatic fever is treated by anit-inflammatory drugs and anitbiotics to control the bacterial population and also iuts effects in causing inflammation of the affctected tissues

Your experimentak reults indicate that antibiotics, such as tetra-cycline, streptomycin, and chloramphenicol, have a broad spectrum of activity againist prokaryotic cells. Why do these antibiotics lack inhibitory activty against eukaryotic cells such as fungi?

Antibiotics are substances that are produced by bacetria and have a static or inhibitory effect on other bacteria. In nature, these compounds have a useful role for their synthesizing organisms in dealing with competing bacterua. Thus, in turn with their natural functions, antibiotics are effective against other bacteria and not on life froms other than bacteria such as fungi or eukarytic

Why are complex media preferable to chemically defined media for routine cultivation of microorganisms?

Artificial media are used for the routine cultivation of microorganisms as the peptones and beef extract are sufficient to provide the nutritional growth requirements for most microorganisms. Thus, knowledge of the specific nutritional needs of the organism is not needed.

Heroin addicts have a high incidence of Clostridium tetani infections. Discuss the reasons for the development of this type of infection in these IV drug users.

Clostridium tetani is an obligate anaerobic bacteria. Deep wounds or skin punctures (caused by using non-sterile needles to inject drugs into skin) usually have anaerobic environment where these bacteria can flourish well (obligate anaerobes thrive well in absence of oxygen). Once bacteria grow and multiply under these conditions, it produces a neurotoxin that can cause severe muscle disorders.

Explain why microorganisms differ in their pH requirements.

Differences in pH requirements among microorganisms are dependent upon the susceptibility of their individual enzyme systems to denaturing at various pH levels.

The physician who treats your puncture wound opts to insert a drain before applying the dressing. What is the rationale for the insertion of the drain?

Drainage of any subcutaneous collections of pus is very important. Fluid accumulation within the tissues affects the wound healing process and increases the rate of infection. Insertion of drain also enables deep penetration of the antibiotics.Therefore, it is important to insert a drain before applying the dressing.

List the three bacertial species in order of their increasing fastidiousness

E. coli, A. faecalis, S. mitis

A household cleaner is labeled germicidal. Explain what that means to you.

Germicidal is referred to as any agent/substance that is capable of destroying germs.The purpose of germicide is to get rid of the area of bacteria and germs that have the potential ability to cause harm to humans and any other types of living things. They are strong agents and toxic to tissues and hence not used on the living tissues. Bactericide is a germicide which kills bacteria. A disinfectant is a chemical germicide which kills wide variety of microorganisms.

Why are crystal violet agar and 7.5% sodium chloride agar considered selective media?

Gram-positive organisms are very sensitive tothe basic dye crystal violet. The exact mecha-nism of action by which crystal violet acts isstill unclear. However, it may be that the dyehas an affinity for the nucleic acids and may in-terfere with reproduction in gram-positive or-ganisms, therefore inhibiting their growth and"selecting" gram-negative bacteria. When in-corporated into a medium, 7.5% sodium chlo-ride agar produces an osmotic environment not conducive for the growth of most organisms other than those classified as halophilic (salt-loving). This medium is excellent for the selection and differentiation of different species of staphylococci, which are halophilic organisms.

Would you expect a heterotrophic organism to grow in an inorganic synthetic medium? Explain.

Heterotrophic organisms require the use of organic carbon sources and, in some cases, organic nitrogen sources and vitamin supplements. These organisms would not grow in an inorganic medium.

You are instructed to grow E. coli in a chemically defined medium containing glucose and NH4Cl as the carbon and nitrogen sourcesand also in nutrient broth that contains beef extract and peptone. Both media are adjusted to a pH of 7. With turbidity as an index for the amount of growth in each of the cultures, the following spectrophotometric readings are obtained following incubation:Based on the above data, explain why E. coli ceased growing in the chemi- cally defined medium but continued to grow in the nutrient broth.

In the chemically defined E. coli culture, which lacks a buffer, growth was terminated due to a radical shift in pH. In the nutrient broth medium, which contains natural buffers, there was no pH shift, and growth was sustained in the presence of adequate nutrients.

Determine the number of PFUs per ml in a 10^-9 dilution os a phage culture that shows 204 PFUs in the agar lawn.

In the plaque forming assay, each of the inefective phage produce a plaque on the host bacterial cell lawn and each of the plaque represents one plaque forming unit (PFU).

In this experimental procudre, why is it important to use a hard agar with a soft agar overlay technique to demonstrate plaque formation?

It is important to use hard agar with an overlay of soft agar as an experimental procedure to demonstarte plaque formation. This is perfromed to obtain a hard base for the host bacterial cell culture to grow as a lawn alon with the viurlent phages for which, the soft overlaid agar provides the optimal growth conditions

Is it possible for thermophilic organisms to induce infections in warm-blooded animals? Explain.

It is not possible for obligate thermophiles toinduce infections in warm-blooded animals because they require temperatures that exceed body temperature for growth. Theoretically, facultative thermophiles can grow at 37°C. However, their optimum growth temperature is 45°C, well above body temperature; therefore, it is unlikely that they would cause infection.

Explain why it is necessary to incorporate buffers into media in which microorganisms are grown.

Microbial metabolic activities will generateshifts in pH within the culture. For example, ifcarbohydrates are primarily utilized, the accumulation of acidic end products will lower the pH of the medium. If protein compounds are metabolized, alkaline end products are released into the environment, thereby raising the pH.

Can the results of a MIC to be used to determine whether an antibiotic is bactericidal or bacteriostatic? If not, set up an experimental procedure to determine whether the effect is bactericidal or bacteriostatic.

Minimum Inhibitory concentration test (MIC): This test is used to measure the lowest effective concentration of antibiotic against the growth inhibition of some specific bacteria. MBC or MLC: minimum bactericidal concentration is referred as the lowest concentration that is required to microbial death of 99.9% of the original inoculum Yes, the results of a MIC test be used to determine whether an antibiotic is bactericidal or bacteriostatic, the antibiotic is going to enter through the process of "diffusion" into the agar from the "disk immersed in antibiotic" placed into the agar form the region of high concentration to region of low concentration. At the time of incubation, the antibiotic is going to emerge into agar in which edge of zone of inhibition depicts MIC, but not the end of diffusion. Kirby-Bauer disk susceptibility test to test effectiveness of bactericidal or bacteriostatic agent: In this antibiotic is placed in disks in nutrient media and tested for the growth of specific antibiotics. The microbe free zones surrounding the wells with antibiotic are taken as criteria to measure the effectiveness.

The release of phage particles from teh host bacterium always occurs by lysis of teh cell and the results in the death of the host. Animal viruses are released by either lysis of teh host cell or exocytosis, a reverse pinocytosis. Regardly of the mechanisms of relase, most infected cells die, while other viruses may escape teh cell without damaging the host cell. Explain.

Most host cells die after viral infections, whike a few might not dies as the infective has thecapacity to leave the host cell without causing damage to the host cell. Enveloped viruses are released from the host cells by process known as "budding". This process does not induce lysis of the host cells, therby damage to the host cell is prevented

Can the disinfection period (exposure) be arbitrarily increased? Explain

No, Toxicity of the chemicals and environment conditions that have to be considered before altering exposure time. Not all microbes are destroyed in the same amount of time.

Will all microorganisms grow optimally at a neutral pH? Explain.

Not all organisms grow optimally at a neutralpH, as their enzyme systems have alreadyadapted for existence in their natural environ-ment. For example, many fungi grow best in anacidic environment, while many species of soilbacteria prefer an alkaline pH.

How does the presence of residential flora influence the infectious process

Residential flora is on a deeper layer and may keep more transient flora on the surface and more spreadable. Residential flora is harder to remove due to its more embedded nature. Normally, residential flora exist symbiotically with your skin, but those who are immunocompromised are at risk of infection. Residential flora can also cause problems if they gain entrance to the host.

How would you record your observation of aplate containing 304 colonies? a plate with 15 colonies

TNTC and TFTC (30-300 is the limit)

Distinguish between F+ and the Hfr bacterial strains

The F factor is extra chromosomal fertility factor which is present in some bacterial strains and such cells are desiganted as F+ cels. The Hfr stains are the high-frequency strains which have reocombined genome consisting of the original genetic content recombines with a part of the genetic content present in the F factor

Explain the signficance of the F factor

The F factor is the fertiliyty factor which is present in the bacterial strain and such cells are designated as F+ cells. The F factor gives a distinction for bcaterial cells that reproduce by sexual mode, through conjugation. During this process of conjugation the F+ ceks transfer the F factor to the F- cells, where teh F+ cells are considered maes and teh F- cells are considered females. Thus, while teh F- cells are recipients the F+ cells are the donors during bacterial conjugation

What are the advantages and disadvanatges of the serial dilution-agar plate procedure?

The advantages: - the enumeration includes only viable cells and non-viable cells are excluded - this approach provides a means to obtain pure colonies of the microbial populations being enumerated for further culturing and analysis The disadvanatges: - requires more time compared to the other methods and also incubation of the plate overnight for the colonies to develop - requires more preparation and material such as glassware adn consumables such as media - can reults in errors in the count due to variations.inaccuracies in the dilution process or the plating process

Explain the chemical methods for measuring cell growth

The chemical methods for measuring cell growth are used to enumerate the microbial numbers indirectly. They utilize the concentration of proteins or nucleic acids in a sample which is in relation to the concentration of the microbes present in the sample.

Why did the most fastidious organism grow poorly in the chemically defined medium?

The chemically defined medium only has certain specific organic and/or inorganic compounds, those compounds were not catering to the nutritional compounds of the most fastidious organism. (*)

Distinguish between dilution and dilution factor

The dilution is defined as teh reduction in the concentration of a sample while the dilution factor gives the extent of dilution as a mathematical umber which is reciprocal to the dilution.

What is the ideal time of the day to perform this procedure? Why?

The ideal time to perform the synder agar test is in the morning before brushing the teeth. This will enable the deetection of the microbes responsible for the formation of caries. This will prevent the loss of the microbes due to brushing or other such activities

Explain how genetic variation may be introduced in eukaryotic and prokaryotic cells.

The major source of genetic variation among prokaryotic cells is by conjugation, transduction, and transformation processes.

Are all members of the resident flora of the mouth capable of initiating dental caries? Explain.

The normally present in resident flora of the mouth can comprise of many types. All these members do not have the capacity to form dentak caries and only those which utilize the carbs present on the surface of the teeth and release lactic acid as a metabolic byproduct can form caries

Your instructor asks you to determine the number of organisms in water sample. Observations of your dilution plates reveals the presence of spreading colonial forms on some of the culture plates. What is the rationale for the elemination of these plae counts in your experimental

The plates with spreading colonies are eliminated from those that are included for taking counts. This is because only plates with discrete colonies are considered for enumeration when using the serial dilution-agar plate procedure. Only discrete colonies give accurate reults either by manual enumeration or by using the Quebec colono counter. Hence, the plates with spreading colonies are not considered for the enumeration of microbial population in the given water sample.

What are the purposes of the indicator strip and the gas generator in the GasPeak system?

The purpose of the indicator strip in the GasPeak system contains methylene blue and changes its color when oxygen is absent. Thus, the indicator strip indicates when the anaerobic condition is established in the culture system. The purpose of the gas generator in the GasPeak system is to produce hydrogen and carbon dioxide gases in the GasPeak culture medium to obtain an anaerobic condition.

How would you explain the mechanisms responsible for the formation of dental caries by resident microorganisms

The resident microbes are the main causative agents for the dental carries. These microbes utilize carbohydrates that are present on the teeth surfaces and in turn produce lactiv acid as a product of their metabolism. The prolonged exposure of teeth to the lactic acid leads to the formation of carries due to the degradation of the dental enamel

Based on your results, what is your tendency to form denal caries? Is this result consistent with your dental history?

The results obtained will be individual specific. A person with a dental history for caries would have more chances of seeing color change after performing the synder test

What is the major disadvantage of microbial count performed by methods order than the serial dillution-agar plate procedure?

The serial dilution agar plate procedure only accounts for living viable cells while other methods count for both living and dead cells.

How would you explain the differential nature of the synder agar medium as used for the detection of the dental caries

The synder agar medium has differential nature that can be used for the detection of dental carries. This is because the agar medium contains a color indicator, bromocresol green that changes its color depending on the change in pH

Why can media such as brain heart infusion and thioglycollate be used for the cultivation of anaerobes?

These media contain meat products that reduce the redox potential of the medium, thus establishing conditions that favor anaerobiosis.

If an organism grew at 20°C, explain how you would determine experimentally whether the organism was a psychrophile or a mesophile.

To determine whether an organism is a psychrophile or a mesophile, cultures should be incubated at 15°C and 37°C. If growth occurs at 37°C but not at 15°C, it is a mesophile. However, if growth occurs only at 15°C, the organism is a psychrophile.

Discuss the effects of the lytic and lysogenic infections on the life cycle of the host cell.

Viruses are classfied as the lytic and the lysogenic forms based on their mode of replication. The lytic viruses infect a host cell, replicate within the host cell and the new virions are released immedfiately after their formation by the lysis of the host cells. Thus, the host cell is lysed in infected by a lytic virus. The lysogenic forms are capacble of remaining within a host and replicate within the host cell without causing immediate lysis of the host cell. The genomes of the lysogenic viruses have the capacity to integrate with the host cell genome and can replicate along with the host cell genome. Thus, a host cell is susceotible for genetic transformation when infected with a lysogenic virus and is not lysed immediately to release the newly formed virions, unlike during the lytic viral

On the basis of the previous data, list the media in order (from best to worst) according to their ability to support the growth of bacteria

Yeast extract broth, nutrient broth, inorganic synthetic broth, glucose broth.

Was the ability of some microorganisms to produce beta-lactamase present prior to their exposure to the antibiotic penicillin? Explain.

Yes ,some microorganisms to produce beta-lactamases present prior to their exposure to the antibiotic penicillin. research revealed that are gentically capable of producing pencillinase.

Explain the purpose of the following:a. Crystal violet in the MacConkey agar medium:b. Blood in the blood agar medium:c. Eosin and methylene blue dyes in the eosin-methylene blue agar medium:d. High salt concentration in the mannitol salt agar medium:e. Lactose in the MacConkey agar medium:f. Phenylethyl alcohol in the phenylethyl alcohol agar medium:

a. Crystal violet in MacConkey agar mediumis an inhibitor to suppress the growth of gram-positive organisms.b. Blood serves to enrich an agar medium to support the growth of fastidious organisms and to differentiate microorganisms, particularly streptococcal species, on the basis of their hemolytic activities.c. The eosin and methylene blue in the EMBagar medium is used to identifyE. coli. The large amount of acid produced by these organisms causes the dyes to precipitate out onto the surface of the colonies, thereby producing a green coloration to the growth. Methylene blue also partially inhibits growth of gram-positive organisms.d. High salt concentration in the mannitol saltagar medium is used to inhibit the growth of or-ganisms other than halophiles.e. Lactose is a major microbial carbon source.In MacConkey agar medium, it serves to differentiate between lactose fermenters and nonfermenters on the basis of their ability to produce acid.f. Phenylethyl alcohol in the phenylethyl al-cohol agar medium partially inhibits growth ofgram-negative organisms; thus, the number andsize of gram-negative colonies is markedly reduce

A soil isolate is found in a basic artificial medium. You suspect that a vitamin supplement is required. A) What supplement would you use to enrich the medium to support and maintain the growth of the organism? Explain.

a. If the organism showed minimal growth ina basic artificial medium, yeast extract could beadded as a supplement, as it contains all the B vitamins.

A soil isolate is found in a basic artificial medium. You suspect that a vitamin supplement is required. B) Outline the procedure you would follow to determine the specific vitamins required by the organism to produce a more abundant growth.

b. To determine the specific vitamin needs ofthe organism, a vitamin assay is required. Inperforming the assay, the control would containall the vitamins. Each of the remaining assayculture media would contain all the vitaminspresent in the control culture with the deletion of one different vitamin from each test tube. Culture tubes lacking growth in the absence of a particular vitamin would indicate that this vitamin is an essential growth factor.

Explain what is meant by plaque-forming units

plaque-forming units are defined as the number of phage particles that are infective, as obtained as a number of plaques that are formed during a plaque forming assay. in the plaque forming assay, each of the infective phage produce a plaque on the host bacterial cell lawn. Thus each other plaque represented one plaque forming unit.

Distinguish between the replicative and maturation stages of a lytic phage infection

the lytic phage infection consists of two distinct stages, the replicative and the maturation stages. The replicative stage is characterized by the replication of teh viral genome using the host cell machinery.

What is the function of the paraffin in this procedure

the paraffin used for performing the synder test is to increase the salivary secretion as can be used as the sample for performing the test

Explain the importance of the streptomycin marker in the parental E. coli strains

the streptomycin marker in parental E. coli strains is important to select recombinant strains that are developed from the parental strains which differ in their streptomycine senstivity. A selectrion medium which utlizes this marker helps in selecting the wild type or the recombinant strains based on the parental streptomycin marker

Discuss the factors responsible for the transformation of lysogenic infection to one that is lytic

viruses are classified as lytic and the lysogenic forms based on their mode of replication. The lysogenic infection can transform into a lytic forma and the factors responsible for such transformatio include certain physical and chemical agents. Examples of these factors are the (UV) radiation and chemical mutagens