Practical Lab 2 Microbiology

You have suspicious that your working place was contaminated with COVID-19. You decide to decontaminate your working table, you look around and found some disinfectants. You need to use the only one which is confirmed to destroy Human Coronavirus. You got EPA registration numbers for all products: EPA Reg. No. 777-89 Lysol® Brand Clean & Fresh Multi-surface Cleaner EPA Reg. No. 11346-4 Clorox QS EPA Reg. No. 9150-2 Anthium Dioxide Please choose the disinfectant which could destroy Human Coronavirus and its time to contact with a contaminated object. (Choose two answers) Hint: use EPA website link and EPA registration number to search: https://www.epa.gov/pesticide-registration/list-n-disinfectants-use-against-sars-cov-2-covid-19

- 2 minutes - Clorox

Based on class data from the table, which bacterium was more resistant and which was less resistant and why? (Select all that apply)

- B. megaterium were more resistant because they form endospores - S. aureus were less resistant because they do not form endospores

Why was half of the plate covered with an index card? (Select all that apply)

- Half of the plate was covered to serve as a control to show that the bacteria were viable before exposure to UV light - Half of the plate was covered to provide an area of comparison to see the amount of bacteria growth when not exposed to UV light and the effects of UV light on the growth

Which of the following are uses for UV light? (Select all that apply)

- air sanitization - sterilize equipment - sterilize work areas - sterilize drinking water and wastewater

Know types of media

-One liquid type of media is broth. -Agar does not melt until it reaches a temperature of about 80°C (176°F). Conversely, once it has melted, it can be cooled to about 45°C (113°F) before it solidifies. In addition, agar has the ability to grow microorganisms over a large temperature range. -Slants are primarily used for storage and transport of microorganisms. slant, a type of solid media. -Plates provide a larger surface area for culturing microbes than slants. Plates are commonly used for isolating microbes. -These mixed cultures must be separated into individual colonies of only one type of microbe to be successfully analyzed. -A pure culture contains only one species of microorganism. Proper use of aseptic techniques prevents pure cultures from being contaminated. -A contaminated culture occurs when unknown microorganisms are inadvertently introduced often via poor technique.

You are asked to perform an acid fast stain. What are the correct steps? Choose the correct dyes and chemicals.

1 Make a smear 2 Heat fix 3 Carbol Fuchsin 4 Acid alcohol decolorizer 5 Methylene blue

How many zones of inhibition have formed on the plate?

2

How many microbes are on the wire loop right now?

500-1000

Bacterium A is [ Select ] . Bacterium B is [ Select ] .

A - non acid fast cocci B - acid fast bacilli

How long does it take to perform and analyze the gram stain?

A few minutes

What is the mechanism by which antiseptic agents show their actions?

All of above - Enzyme inhibition - Cell wall destruction - Damage to the DNA

Which of the following can be an active agent in an antiseptic?

All of above -Alcohols -Iodine -Phenols

Why is the bacterial cell wall of such great importance?

All of the answers are correct -The cell wall is involved in inducing disease in the host. -The cell wall is essential for bacterial viability. -Antibiotics can target the cell wall.

What is aseptic technique?

Aseptic techniques are the techniques needed to prevent the accidental or inadvertent spread of microorganisms beyond the intended working environment. 1. Hands must be washed, work surfaces must be cleared of unnecessary items, and sterilized with a 10% bleach solution or a bleach-based cleaner before beginning the Experimentation. 2. The equipment used to isolate cultures has to be sterilized. Either an open flame or isopropyl alcohol may be used to eliminate contamination that is naturally picked up from the environment. In campus laboratories, equipment is often sterilized with an autoclave or micro-incinerator. 3. When opening cultures or sterile media: Work quickly and efficiently. Do not talk or breath over the culture. When tubes and vials are open, keep them tilted away from the mouth and nose. Never lay caps or lids on a bench or work area, as these areas most likely have contaminants. When working with a culture, ensure the instruments used are sterile. this will include inoculation loops and swabs. Sterile instruments are provided by Hands-On Labs, Inc. (HOL) You must work to maintain the instruments' sterility 4. Some general precautions include the following: Avoid producing aerosols (anything that can be introduced into the air). This includes avoiding splashing when pipetting media and inoculating cultures. Keep the lid to the culture closed whenever possible. Incubate plates inverted (agar-side up). Agar contains a lot of water. When plates are incubated with the lid side up, water will condense on the lids; this condensation can drip back onto the agar plate and possibly contaminate and/or smear the results. Always write on the bottoms of the plates, not the lids. This will prevent unlabeled plates if lids are misplaced. When not in use, tubes and plates should be properly stored away from children and pets. All equipment and work surfaces should be cleaned with a 10% bleach solution or bleach based cleaning product at the end of the experiment.

Which circle contains bacteria?

B contains bacteria

How long did it take the UV light to completely kill B. megaterium?

B. megaterium were never killed in this experiment

It seems that the new infection has a different form of the transpeptidase enzyme that penicillin can't bind to - what a sneaky resistance mechanism! What is one other way suggested by theory by which a previously sensitive bacterial strain can become resistant to penicillin?

Bacteria can express an enzyme capable of inactivating penicillin before it can bind to transpeptidase

How long did it take for the UV light to completely kill S. aureus?

Between 8 and 16 mins

We've got a probe and a kidney dish. The probe was used to explore the patient's teeth, so will have contacted the patient's mucous membranes and the outside of their teeth. What classification would you give to the dish and the probe if they were used as per their normal intended processes?

Both would be semicritical

Matching

Capsule An outer covering on some species of bacteria that consists of polysaccharides or polypeptides. Structural staining A staining process that dyes certain structures in a cell a specific color so that it can be identified. Endospore A dormant structure that is produced inside some species of bacteria when conditions become unfavorable. Virulence A measure of the severity of a disease that a microorganism causes. Mycolic acid A lipid contained in the cell wall of acid-fast bacteria. Differential staining A staining technique that uses more than one stain, which is then used to group bacteria into a specific category Primary stain The first stain used in staining techniques that require the use of more than one stain. Counterstain The final stain used in staining techniques that require the use of more than one stain.

Is there anything else that might help them?

Chlorhexidine is an effective antiseptic at the right concentration, maybe they could try this as a mouthwash?

You will see values on the left and have to select the matching value on the right from a dropdown.

Disinfectants The chemical agents designed to inactivate or destroy microorganisms on inert surfaces. Decontamination The process of cleansing an object or substance to remove contaminants such as micro-organisms or hazardous materials, including chemicals, radioactive substances, and infectious diseases. Sterilization Any process that removes, kills, or deactivates all forms of life and other biological agents like prions present in a specific surface, object or fluid. Antiseptics An antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction. Denaturation A process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state.

A broad-spectrum disinfectant will kill all germs on hard surfaces.

False

For the most accurate results always make smears and stain cultures that are between 24 - 48 hours old when performing the endospore stain.

False

The capsules of bacteria can be observed in gram-stained smears.

False

Under de-colorization would result in all bacteria appearing gram negative and over-decolorization would result in all bacteria appearing gram positive.

False

Where do we find the Teichoic acids?

Gram positive bacteria

What is the outcome of a well executed Gram Stain?

Gram positive will appear purple, and gram negative will appear pink

What does the graph represent?

Logarithm of bacteria on the wire loop

The drug you added seemed to prevent cell growth much quicker, but the cells didn't burst like before. According to the theory, why do you think clindamycin and metronidazole produce a different effect to penicillin?

Metronidazole targets the DNA of microbial cells and clindamycin targets the ribosome. neither of wich create a lytic effect like penicillin.

Even if resistance was not an issue, antimicrobials would only have a discreate range of bacteria that they can exert their effects on. Why do you think this is?

Microbial components vary over different species and strains. If the organism naturally doesn't express the target component, the drug won't be able to exert an effect on it.

What unique properties do acid fast organisms possess that contribute to their virulence?

Mycolic acids

According to the theory, how does penicillin kill bacteria?

Penicillin targets a cross-linking reaction in cell wall manufacture. By blocking the enzyme responsible, penicillin stops the bacteria from producing new peptidoglycan.

Why do you think penicillin has failed to destroy the bacteria we have cultured from the patient's sample?

Perhaps a few cells from the old infestion developed resistance and so weren't cleared by the previous treatment. They could have gone on to develop this second abscess.

Why do you sterilize the loop between streaking each quarter of the plate?

Reduce the number of bacteria on the wire loop

What species are we dealing with?

Salmonella

What are the black and red antibiotic resistant species growing on the plate?

Salmonella and E. coli

Which of the following is not susceptible to alcohol compounds?

Spores

Based on your explorations and theory reading in the lab today, which sterilization method do you think would be best for these items?

Steam and high pressure will kill everything; let's use the autoclave.

You will see values on the left and have to select the matching value on the right from a dropdown.

Sterilant an agent that destroys all forms of microbial life Sanitizer an agent that reduces but may not eliminate microbial number to a safe level Disinfectant an agent that kills or inhibits growth of pathogenic microbes and is used only on inanimate objects Antiseptic an agent that kills or inhibits growth of pathogenic microbes and is sufficiently nontoxic to be applied to living tissues

Based on your exploration and the theory, what do you think the difference is between decontamination and sterilization?

Sterilization is a type of decontamination, but not all decontamination techniques result in true sterilization.

What do we highlight when we talk about antimicrobial resistance?

The ability of a microbial strain to withstand the effects of a previously effective antimicrobial agent.

When we say an antimicrobial agent exhibits 'selective toxicity', what do we mean?

The agent is only toxic to microbial cells because it targets a cell component only found in microbes and not present in the host organism

Where are the Lipopolysaccharides found?

The gram negative outer membrane

Why do we need a positive control in this experiment?

The hypochlorite is known to cause inhibition, so shows inhibition can occur in the current experiment and what it looks like; this is the positive control.

What tentative interpretation can you make from what you observe?

The penicillin has failed to make a zone despite the positive control's success. The bacteria may be resistant to penicillin.

According to the theory, why is it important to know how much of a pathogenic risk an item poses to an end-user?

The risk determines how you must decontaminate an item so that the next user doesn't get infected.

How does wet heat sterilization work to inactivate bacteria and other infectious pathogens?

The sustained heat and pressure denatures proteins, without proteins nothing can replicate or remain infectious

What is the gram reaction of the bacteria in the Cerebral spinal fluid?

They are gram negative

How does a capsule contribute to pathogenicity?

They can prevent the bacteria from being phagocytized

Why did you sterilize the wire loop?

To avoid contamination

Why are the bacterial samples stored in the fridge?

To reduce bacterial growth

Why do we use positive and negative controls in the gram stain?

To verify that the gram stain was performed properly.

If old bacterial cultures are used for gram staining the cell walls may be broken down/weak and result in gram positive bacteria staining as gram negative.

True

The plates that had the lids left on showed that UV light cannot penetrate through the clear plastic of the petri plate lid.

True

You need to let a disinfectant remain wet on a surface before wiping it dry.

True

What disease(s) are diagnosed using the Acid Fast Staining procedure? Note: More than one question may be correct.

Tuberculosis Leprosy

What happened between the two timepoints indicated by the blue lines?

Two bacteria cells were transferred to the agar plate

Match the term to its defintion:

Ultraviolet germicidal irradiation - This is a method of disinfecting that uses short-wave UV light to kill bacteria by disrupting their DNA. Thymine dimer - A molecule consisting of two linked thymine subunits that are formed in DNA by a covalent linkage, in response to ultraviolet radiation. They can sometimes be excised by repair enzymes, though the process sometimes makes errors that create mutations. Germicidal UV - Short range UV (UVC), is mutagenic to bacteria, viruses, and other microbes. Mutation - Changes to DNA that are not repaired and may lead to death of the organism. Staphylococcus aureus - Gram positive, non spore-former Bacillus megaterium - Gram positive, spore former

The standard method we could use to compare disinfectants is called the __________ method.

Use-Dilution

How did you get on with the tools? How did you process them?

We cleansed them using biological detergent then rinsed and dried everything before autoclaving.

Did you learn anything about bacteria causing the infection?

We conducted a diffusion disc assay, the new infection was not inhibited by a penicillin challenge. Maybe the new infection is penicillin-resistant?

Now that you've explored the lab and found what you've got on hand to work with, what do you think the first step should be for decontamination these tools?

We should remove loose soil and debris by washing using a biological detergent.

Is the negative control properly stained?

Yes, it appears gram negative, with the bacteria stained pink

Is the positive control properly stained?

Yes, it appears gram positive, with bacteria stained purple

The shape of the gram negative bacteria shown here is?

bacilli

This is a capsule stain. The capsule stain procedure involves [ Select ] to stain the bacterial cell and [ Select ] to stain the background. This makes the capsule appear as [ Select ] . It is important when preparing this slide that the slide is not [ Select ] otherwise the capsule will be destroyed.

direct staining negative staining an unstained halo fixed

Assume you stain Bacillus by applying malachite green with heat and then counterstaining with safranin. Through the microscope, the green structures are:

endospores.

Which cell wall component can cause toxic shock in the host?

lipopolysaccharides

Which of the following refers to a germicide that can kill vegetative cells and certain enveloped viruses but not endospores?

low-level germicide

What is the gram stain method?

staining method used to differentiate bacterial species into two groups

In the disk-diffusion assay, a large zone of inhibition around a disk to which a chemical disinfectant has been applied indicates ______________ of the test microbe to the chemical disinfectant.

susceptibility ALSO POSSIBLE ANS: weakness sensitivity vulnerability

The red bacilli are the [ Select ] . The green ovals are [ Select ] .

vegetative cells endospores