Lab Exam II: 22, 23, 24, 25,26, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45.

Killing by UV light procedure

1. divide each plate in half (mark the bottom of the plate. 2. Lavel one side of the plate "covered" and the other side "uncovered." 3. Label one plate A one B and one C 4. Use a loop to streak each sample of bacteria on half of each plate 5. Place plate A under the UV lamp. Remove the lid from the plate and cover half of the plane with an index card. Turn on the lam and expose the plate for 30 secs. Make sure you are wearing goggles and to not have your dads under the lamp. 6. Expose plate B for 1 minute. 7. Expose plate C for 5 minutes 8. Put the lids back on your plates and put the plates in the incubator lid side down. 9. the incubator must be dark (to prevent light repair), and you should put the plates into the incubator as soon as possible.

The reaction of catalase is

2H2O2 ----> 2H2O + O2

antibiotic

A chemical secreted by a living organism that kills or reduces the reproduction rate of other organisms

What is an Antibiotic?

A type of antimicrobial (chemical substance) that is made by microorganisms to inhibit or destroy the growth of other microorganism Antibiosis: literally means "against life"

antimicrobial

An agent that kills or inhibits growth and reproduction of microorganisms.

killing by UV light which time was the most effective? Least effective?

Both Serratia and Bacillus was killed under the UV light for 5 minutes, the longer the exposure the most damages the UV light can cause to the DNA of the bacteria. Serratia was sensitive to the 1 minute exposure under the UV light, but the bacillus was less sensitive to the 1 minute exposure of UV light since we used bacillus spp 1-week -old that had formed an endospores

Medical Application

Catalase activity is very useful in differentiating between groups of bacteria For example, the morphologically similar -Enterococcus (catalase negative) and -Staphylococcus (catalase positive) can be differentiated using the catalase test

What is catalase?

Catalase is an enzyme found in the cells of many living tissues Speeds up a chemical reaction which breaks down hydrogen peroxide, a toxic chemical, into 2 harmless substances 1. water and 2. oxygen

What is an antiseptic?

Chemical that can be used on the skin and mucous membrane which can able to destroy or inhibit most pathogens

as we progress in the field of medicine out technology keeps more people alive with immunosuppressive conditions (transplant recipients, cancer chemotherapy, HIV/AIDS, or old age)

Debilitated and immunosuppressed individuals are more likely to develop infections caused by many opportunistic pathogens. Those pathogens may have been acquired from another person or may have already been in or that individual.

What is an UV Light?

Energy reaches earth's surface is visible light A type of electromagnetic radiation Wavelength shorter than X-ray ~300 to 400 nanometers of radiation

Oxidase Medical Application

For the identification of bacteria that produce Cytochrome c Oxidase Useful procedure in the clinical laboratory gram negative pathogenic species of bacteria such as: Vibrio Pseudomonas aeruginosa Neisseria gonorrhoeae Moraxella

What is an UV light? Two forms of radiation used to kill microbes

Gamma Radiation - Toxic, because it create free radicals when colloid with molecules Ultraviolet Radiation - Kills the microbes by damaging the DNA

With which antibiotic should PT B be treated.

Gentamicin since it was the only one the kill or inhibited the growth of the Pseudomonas.

it is estimated that students and other humans shed 1.5 million skin cells per hour. What problems might that create in the microbiology lab or surgical suite?

If we don't wash our hand all the skin will contaminated and spread normal biota, since the shed skin also contain bacteria. In the surgical suite it can be fatal for the PTs.

Media used for the purpose of cultivating fastidious organism--grow when specific nutrients are included.

Media supplemented with highly nutritious materials such as blood, serum, or yeast extract. EX. blood agar, chocolate agar

we have normal biota (microbes) on our skin

Micrococcus luteus Staphylococcus epidermidis Staphylococcus aureus Corynebacterium propionibacterium acnes Normal biota are harmless and some are even beneficial. They are usually difficult to remove from the skin.

How antibiotics works?

Mueller-Hinton Agar Plate Organism Antibiotic Disc Zone of Inhibition

K-B test involves special type of agar

Muller-Hinton agar, which must be poured to a standard thickness. The plates re inoculated with cotton swabs rather than inoculating loops.

Killing by UV light

OBJECTIVE Determine the advantage and disadvantage of using UV light as a sterilizing agent

there are different forms of cytochrome oxidase, depending of the bacterium

One form s called cytochrome c oxidase. the purpose of this test it to determine if the bacterium if interest has cytochrome c oxidase in its electron transport chain.

What is Hand Washing?

One of the best practice to limit the transmission of pathogens Three important components: 1. Detergents: Emulsify the skin oil to remove bacteria 2. Friction: Physically remove the bacteria 3. Antiseptic: Kills the remaining organisms without harming skin.

What is Oxidase?

Oxidase is an enzyme plays an important role in the ETC during aerobic respiration Cytochrome c Oxidase reduces O2 to form H2O

which patient's infection appear to be the most difficult to treat? Why?

PT B because most of the antibiotics did not kill or inhibits the growth of the Pseudomonas, so they are resistant to Pseudomonas infection, It was only sensitive to Gentamicin.

Which patient's infection appears to be the easiest to treat? why?

PT S because all the antibiotics kill and inhibited the growth of the bacterium, since Staphylococcus infection is more sensitive to most of the antibiotics.

Oxidase Mechanism • If Cytochrome c Oxidase is present it oxidizes the reagent to purple color Negative • If the enzyme is not present, the reagent remains reduced and colorless

Positive • If Cytochrome c Oxidase is present it oxidizes the reagent to purple color Negative • If the enzyme is not present, the reagent remains reduced and colorless

Which is more likely cause infections of deeper, less oxygenated tissue: a member of the enteric or Pseudomonas? Why?

Pseudomonas eruginosa, which utilize cytochrome c oxidase, aerobic gram-negative is a oxidase-positive. This causative bacteria frequently exhibits antibiotic resistance. Also shown the ability to grow on a surprisingly wide range of materials, including iodophor antiseptics, exfoliative sponges.

catalase negative: Complete lack of bubbles indicates a negative reaction.

Result: No bubbling on slide, no reaction of H2O2 Interpretation: Catalase-negative organism

Organisms used in UV test

Serratia--spore formation bacteria Bacillus cereus (use s culture older than 1 week)

What would you expect to happen if you forgot to remove the lids on all your plates when you put them under the UV lamp? Why?

The UV light will not damages the DNA and the microorganisms will grow. Since, UV light will not go through plastic, glass, and many other materials.

you were distracted by another student in the classroom and forgot about you oxidase test for 15 minutes. When you check your test result, you notice a dark blue ring, but no added coloration to the colony smear. What can you conclude from this result?

The faint blue-purple ring around each organism in the photograph on the right is caused by the reaction of the oxidase reagent with the paper.

the most effective hand washing should include three components

The first: soap or detergent, which are produced by a process called saponification, which involve treating a fat with alkali (base) in the presence of heat. It emulsify the skin oils in which bacteria is trapped (especially between the deep dermal ridges on our hands) This ability to remove dirt (including bacteria) from surfaces is why detergents can also be referred to as surfactants. it are amphipathic, which means that they have a lipid-soluble (fat-soluble) portion and a water-soluble portion. They can disrupt the lipid portion of cell membrane. The second component is friction. it helps us physically remove the emulsified skin oil with the trapped bacteria. The third component is incorporation of an antimicrobial agent called an antiseptic in to the soap. washing your hands with soap or a detergent helps to remove transient microorganisms, but your normal biota is usually not completely removed

Disadvantages of UV light

The major disadvantages are: - Poor penetration - Damaging effects seen over long exposure to human tissues - Retinal damage - Cancer - Skin wrinkling

Application if UV light

Ultraviolet radiation is a powerful killer of: Fungal cells Spores Bacterial cells Protozoans Viruses Used for disinfection but not sterilization. Used in germicidal lamps in hospital rooms, operating rooms, food preparation areas, and dental offices.

TSA plates

Used to compare abundance and diversity of microbes before and after hand washing

Mueller-Hinton Agar Plate

What type of media was used for the Antimicrobial sensitivity test?

How UV light works?

When DNA molecule absorbs the UV light the bonding relationship in the molecule affected Formation of DIMERS (thymine) That leads to mutation UV light ~200 and 290 nm is most lethal

Tips for succes for oxidase test

When obtaining results, look for a blu-purple color change concentrated where the cells were added to the paper. As the reagent sits on the paper towel, it forms a blue ring, which DOES NOT indicate the presence of cytochrome c oxidase. This test must be done with cultures growing on solid media.

as the bacteria cell carries out different metabolic pathways

a harmful by product called hydrogen peroxide (H2O2) is produced, is produced by the electron chain during the reduction of oxygen, O2 acts as the final electron acceptor in the process of aerobic respiration

in order to test for the presence of cytochrome c oxidase enzyme

a reagent called oxidase reagent is added to the cells of interest.

Oxidase

bacterial colonies having cytochrome oxidase develop blue color

When DNA absorbs UV light, in particular UV-C

bonding relationships within the molecules can be affected. A major ex. is the formation of thymine or cytosine DIMERS (bonds between two adjacent pyrimidines in the DNA strand). These can lead to mutations

nucleotides are the

building blocks of nucleic These subunits can serve as nutrients of the bacteria

By detoxifying H2O2

catalase allows the bacterium to survive and thrive in oxygen-rich environment.

UV light can cause

cataracts or other eye damage.

Exposure to a large amount of UV light

cause more damage than cell systems can repair, leading to death of the cell.

The reagent oxidase is a redox indicator that is

colorless when reduced dark blue purple when oxidized The oxidase reagent will donate electrons to cytochrome c oxidase if it present By donating electrons the reagent is oxidized and turns purple. This color change is an indication that the organism produces cytochrome c oxidase as part of its electron transport chain.

in the process of cellular respiration, high potential energy molecules like glucose are

completely oxidized. These electrons are then shuttled to the electron transport chain located inn the PLASMA membrane of the bacterium.

as these redox reactions are taking place

energy is released, which is ultimately used to produce ATP.

DNase is an

extracellular enzyme,

The two form of radiation that are used to kill microorganism are

gamma radiation--AKA as ionizing radiation--has it greatest impact when it collides with molecules in the cell, creating free radicals, which are toxic. ultravioleta radiation--AKA as nonionizing radiation--prinarily kills by damaging DNA.

the UV light emitted by

germicidal lamps is valuable for killing microbes in the air and on surfaces such as inside transfer hoods for tissue culture, clean rooms, and operating rooms.

The catalase test is useful in distinguishing

gram-positive catalase cocci from one, another. -Staphylococcus --infection can sometimes be difficult to treat. -Micrococcus Gram-negative catalase -Enterococcus--great concern as cause of hospital-aquired infections due to its natural resistance to multiple antibiotic, -Streptococcus--infection can sometimes be difficult to treat.

Catalase reaction is important to cells becuase

hydrogen peroxide (H2O2) is produced as a byproduct of many normal cellular reactions.

UV light with a wavelength between 200 and 290 nanometers (nm)

is the most lethal, because these are the wavelengths that are absorbed the most by DNA.

In order to detoxify this ROS

many bacteria produce the enzyme CATALASE.

Microorganism are also involving

new mechanisms of pathogenicity and antibiotic resistance.

In the most natural habitat most bacteria are

not pathogenic. As matter fact, only one in 50,000 bacteria can cause disease.

Oxidase results

organism C produces cytochrome c oxidase as indicated by the color; Organisms A and B do not produce cytochrome c oxidase. The faint blue-purple ring around each organism in the photograph on the right is caused by the reaction of the oxidase reagent with the paper.

Enterococcus its natural resistance to multiple antibiotics including

penecillins cephalosporins sulfamethoxazole -and its more recent accession of resistance to vancomycin or fluoroquinolones. -Treating hospital-acquired Enterococcus often requires newer antibiotics or combinations of antibiotics.

UV light will not go through

plastic, glass, and many others materials. because of this, it is mainly used for killing microbes on surfaces, in air, or in water.

Did you see a difference between the survival of the two types of bacteria when exposed to UV light for various times? What do you think is the reason?

plate A was exposed to the UV light for 30 seconds, which did not was time enough to penetrate the microorganisms to damages the DNA. Plate B was exposed to the UV light for 1 minute time enough to damaged the DNA of Serratia microorganism but not enough to damages the DNA of the Bacillus, since it was a in endospore formation. Plate C was exposed to the UV light for 5 minutes time enough to damages the DNA of both microorganisms.

One physical method that can be used to control microbial growth is

radiation

The function of the cytochromes in the electron transport chain, through a series of

redox reactions, is to transfer electrons and reduce oxygen in the last step.

Enzymes of the electron transport chain called oxidase

reduce oxygen to form, among other products, hydrogen peroxide (H2O2), which is considered a REACTIVE OXYGEN SPECIES (ROS) and can cause significant damage to important molecules of the bacterium like DNA, RNA, and proteins.

catalase positive: Bubbles should appear after placing H2O2 on the slide.

result: bubbling due to the production of oxygen from the breakdown of H2O2 Interpretation: Catalase-positive organism

The UV light is very damaging to the eyes

so must be used when people are not present or use eye protection during exposure.

what effects did you see from the use of an antiseptic rather than soap?

soap removes microorganisms as well as other debris to reduce contamination to safe levels The antiseptic lowest level of antimicrobial control; reduces microbes on human skin

the K-B test method is widely used for

testing the sensitivity of bacteria to antimicrobials. 1. the test is done by first covering the surface of the plate with the bacteria being tested. 2. paper discs containing known concentrations of different antimicrobial are then placed on the surfaces of the plate. 3. the concentration of the antimicrobial compound found in the disc are the same as the effective concentration found in the body someone taking that antimicrobial. 4. The antimicrobial agent in the disc diffuse out of it, forming a concentration gradient in the agar around the disc. 5. the concentration of the antibiotic at the age of the disc is high and gradually diminishes as the distance from the disc is high and gradually diminishes as the distance from the disc increase to a point where it is no longer inhibitory for the organism, which then grows freely. 6. if the antimicrobial inhibits the growth of the organism, a clear circular area appears around the disc. 7. this is called the zone of inhibition. 8. the method for doing the K-B test is highly standardized for use in clinical laboratories.

What organism is PT G apparently infected with?

the PT apparently is infected with Micrococcus luteus, since the sample bubbles after putting H2O2 indicating a reaction for catalase-positeve

Which which antibiotic should PT X be treated?

the PT can be treated with Tetracycline or Chloramphenicol, since both were kill or inhibit E. coli.

If the cells did not break down the hydrogen peroxide

the cells would be poisoned and die.

the biochemical process of hydrogen peroxide detoxification by aerobic bacteria by the production of

the enzyme catalase

Once the DNase extracellular enzyme is secreted

the enzyme diffuses into the environment and breaks down macromolecules to the level of the mono nucleotide or polynucleotide

cytochrome oxidase is

the final enzyme of the electron transport chain that reduces oxygen to form H2O

Antimicrobial susceptibility testing

the goal is ti find out whether the bacteria being tested are SENSITIVE to the antibiotic (will be killed by it) or resistant to it (will be ineffective for treating the PT)

oxidase procedure is used to distinguish among

the gram-negative rods, some of which can cause corneal infections. members of the enteric, which are facultative anaerobes, do not utilize cytochrome c oxidase and are considered oxidase-negative. Aerobic, gram-negative Pseudomonas aeruginosa is oxidase-positive.

what difference did you see between the unwashed has washed skin? between the use of a scrub brush and no scrub brush?

the plate with the sample of the unwashed hands show the norma biota of the skin. in the washed skin we see less colonies of the normal biota of the skin. the hands that were scrub has lest colonies than the ones were not scrub.

the function of catalase is

to convert H2O2 into H2O and O2 gas, which are harmless to the cell.

Those normal biota are in competition with

transient organisms that are found on the skin for short periods of time but do not grow there. Transients are acquired by contact with contaminated surfaces and are usually easily removed. Some transients cans cause disease. we can easily transfer transient organism from one person to another.

bacteria and other microorganism are

ubiquitous (they are everywhere). they are in the air we breathe, the food we eat, and the water we drink.

Steps for using a swab to make a lawn of bacteria

use your swab to make parallel streaks in one direction that are close together. Rotate the plate 90 degrees and make another set of parallel streaks. Rotate the plate 45 degrees and make a final set of parallel streaks.

DNase (extracellular enzyme) is produced

within the cell and secreted outside of the cell wall.

What is Kirby-Bauer Method?

• Developed in the USA in 1966 • Based on NCCLS ( National Committee for Clinical Laboratory Standards ) data • For testing the bacteria to antimicrobials

Oxidase Procedure

• Draw two circles on the paper towel • Add oxidase reagents to each circle • Use the tooth pick to add the organisms 1. E.coli 2. Micrococcus luteus • Use enough bacteria to visible on the paper • You can see the reaction within 1mt

Mechanism of catalase test

• If the enzyme is present it breaks down peroxide into water and oxygen releasing strong bubbles - Positive • No bubbles - Negative

Kirby-Bauer Method application

• K-B Test is routinely done to monitor the prevalence of antibiotic resistant bacteria • Observe for a trend in order to take precautionary measures - For example: • development of new drugs • determining the resistance and modify existing drugs accordingly

Procedure of K-B test

- Prepare a pure culture (18-24 hrs) of the sample on a non-selective medium - Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standard *sample = 0.5 McFarland standard Within 15 minutes of adjusting the turbidity • dip a sterile cotton swab into the sample • streak a lawn of bacteria on Mueller-Hinton agar *Leave the lid agar for 3-5 minutes (no more than 15 minutes) to allow plate to dry *Label 3 Mueller-Hinton Plates • Use a sterile swab to make a lawn of bacteria using the following organisms 1. Escherichia coli 2. Staphylococcus aureus 3. Pseudomonas aeruginosa • Use a disc dispenser and place 6 different antibiotic discs around the medium.

to determine if an organism produce catalase

1. An inoculum is placed on a glass slide. 2. A few drops of H2O2 are then dispensed onto the cells. 3. If the organism produce catalase, bubbling will appear due to the production of O2 gas from the breakdown of H2O2 and the organism is considered Catalase-Positive.

Oxidase Organisms needed

1. Escherichia coli 2. Micrococcus luteus

Killing by UV light Materials Required

1. Goggles 2. UV lamp (254 nm wavelength) 3. Marker 4. 3 x 5 index cards or half-sheets of paper 5. Box or shield to cover lamp 6. Nutrient agar plates (3 per team) 7. Swab 8. Microincinerator

Procedure of hand washing test

1. Label 4 plates

Organisms needed for catalase test

1. Micrococcus luteus (+) 2. Enterococcus faecalis (use pipet) (-) 3. Patient G Culture (+)

Materials Required for catalase test

1. Microscope slide 2. 3% H2O2 solution 3. Inoculating loops 4. swabs

Materials Required for K-B test

1. Mueller-Hinton plates (3 per team) 2. Sterile swabs 3. Metric rulers (1 per team) 4. Chloramphenicol (30 ug), Erythromycin (15 ug), Kanamycin (30 ug), Penicillin (10 ug), Streptomycin (10 ug), Tetracycline (30 ug) 5. Tweezers 6. 250 ml beaker with 70% alcohol

Oxidase Materials Required

1. Paper towel 2. Toothpicks 3. Oxidase reagent dropper (BBL)

Organisms used for K-B test

1. Patient B - Staphylococcus aureus 2. Patient S - Pseudomonas 3. aeruginosa Patient X - Escherichia coli

Killing by UV light Organisms required

1. Seratia (24- to 48-hour broth cultures) 2. Bacillus (4 week old broth culture)

Materials Required of Hand washing test

1. Sterile swabs 2. Inoculating loops 3. Tube of sterile water (4 per team) 4. Sterile surgical scrub brush (1 per team) 5. Regular soap (nonantibacterial) 6. Betadine or other antiseptic 7. TSA plates (4 per team plus 2 extra per class)

Procedure for catalase test

1. Use the swab to scrap some colonies 2. Add the cells to the glass slide 3. Add 2 drops od H2O2 onto the cells 4. Watch for bubbling as an indication of oxygen production 5. Discard the used slide in the sharp container