Micro Lab Final

hospital acquired infection

An infection occurring in a patient in a hospital or healthcare setting in whom the infection was not present or incubating at the time of admission, or the remainder of an infection acquired during a previous admission; See nosocomial infection

List three different types of disease transmission methods and give an example of each

Direct contact- person to person, droplet Indirect contact- airborne, contaminated objects food and drinking water, animal-to person contact, animal reservoirs, insect bites vectors, environmental reservoirs

Which organisms did not grow on MSA plate?

E.C and SM

Explain the purpose of eosin and methylene blue dyes in eosin-methylene blue agar

EMB is a selective differential medium used to isolate fecal coliforms. Eosin and methylene blue are pH indicatore dyes which combine to form a dark purple preciptate at low pH they alse inhibit the growth of most gram positive organisms

indirect contact

Exposure or transmission of disease from one person to another by contact with a contaminated object.

did you need the oil immersion to observe the shape of eukaryotes?

No because eukaryotic cells are larger

do all the bacteria samples grow on EMB and MacConkey agar?

Not all of them should grow on EMB and MacConkey agar because they are selective and differential medium

you observed unstained and stained yeast specimen. Which one was easier to observe? explain the reason

The stained one because the staining makes the eat easier to see

MSA plates

Trypticase soy agar or tryptone soya agar are growth media for the culturing of bacteria. They are general-purpose, nonselective media providing enough nutrients to allow for a wide variety of microorganisms to grow.

bacillus

Rod shaped bacteria

staphylococcus

a genus of gram-positive bacteria that are potential pathogens, causing local lesions and serious opportunistic infections

broth medium

a liquid medium that lacks a solidifying agent

why the capsule staining is called a negative staining method?

a negative stain does not stain cells it stains the background of the cells

illuminator

a steady light source used in place of a mirror

Describe the difference between a wet mount slide and simple staining

a wet mount allows one to observe bacteria in terms of the motility and provides some insight on the organisms overall morphology. staining makes bacteria more easily seen and it allows their morphology to be visualized easily

negative stain for capsule (special stain)

acidic dyes such as eosin and nigrosin are used to stain the background and basic dyes are used to stain cells. the capsule is not stained but can be seen as an unstained layer in contrast to the stained cell and background background-stained pink cells- stained blue capsule- unstained

alpha hemolysis

alpha or partial hemolsis is demonstrated by organisms such as streptococcus pneumonieae which can break down RBC only partially. the colonies are greenish-yellow and discoloration of agar is observed on plates (think about apperance of your sputum when you are sick with infection)

nosocomial infection

an infection acquired during hospitalization

Acid-Fast stain for mycobacterium and nocardia

bacteria containing waxy substances in their cell walls do not stain well using gram staining. Heat/steam is used to stain waxy cell surface using carbolfuchsin. A mix of acid-alcohol is used to decolorize cells that do not have waxy cell surface and then counterstained with methylene blue. bacteria contianing waxy cell surface- stained red non-acid fast cells- stained blue

explain the purpose of lactose in MacConkey agar

because it can identify bacteria based on their ability to ferment lactose. Lactose provides a source of fermentatable carbohydrates allowing for differentiation

Why is mannitol salt agar used as a selective medium for normal skin microbiota?

because most bacteria on the skin are salt tolerant and will most likely grow on a MSA plate

streptococcus

berry-shaped (bacterium) in twisted chains

beta hemolysis

beta or complete hemolysis bacteria like streptococcus pyogenes completely digests RBCs creating a clear sone around the colonies

Is blood agar selective or differential?

blod agar is differential bacteria are identified based on hemolysis

What is the purpose of flaming the inoculating loop or needle before and after each inoculation

by flaming the inoculating loop you are sterilizing it preventing any unwanted contamination of specimens

selective toxicity means

drug is very toxic to the microorganism but not to human cells

give two examples of gram negative bacteria

e. coli serratia marcesens

Schaffer-fulton endospore stain

endospores are impermeable to most chemicals. Therefore, steam is used to drive the primary stain, malachite green into the endospore. After decolorizing, vegetative cells are counterstained with safranin. endospore-stained green vegetative cells- stained pink

Gamma hemolysis

enterococcus faecalis with gamma or no hemolysis does not digest RBC so agar is unchanged and no lysis occurs

direct contact

exposure or transmission of a communicable disease from one person to another by physical contact

diaphragm or iris

many microscopes have a rotating disk under the stage. This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide

BAP plates

ontain mammalian blood (usually sheep or horse), typically at a concentration of 5-10%. BAPs are enriched, differential media used to isolate fastidious organisms and detect hemolytic activity.

diplococcus

pair of cocci

aerosal transmission

person-to-person transmission of pathogens through the air by means of inhalation of infectious particles. Particles up to 100 μm in size are considered inhalable (inspirable)

List three factors the protect the skin from infection

sebum salts overlapping dead skin cells in dermis

Describe the purpose of specialized media and differential media

selective media -designed to grow specific groups of bacteria; used for the primary isolation of a specific organism or group of organisms from mixed samples that contain many different bacteria; can be used to inhibit the growth of normal flora so pathogens can be isolated differential media-designed to show visible differences as bacteria grow on the media; not all bacteria have the same enzymes; the ability to ferment a sugar or use an amino acid can help identify the organism; variations on differential media are due to the way bacteria react to the chemicals in the media (bacterial enzyme --> biochemical reaction --> color change)

describe the difference between simple staining and differential staining principles

simple stains use only one type of stain to color microorganisms differential stains use two or more different types of stains to show different parts of the microorganism

fine adjustment knob

small, round knob on the side of the microscope used to fine-tune the focus of your specimen after using the coarse adjustment knob.

Cocci

spherical bacteria

spirilla

spiral shaped bacteria

give two examples of gram positive bacteria

staphylococcus aureus streptococcus pneumonia

arm

supports the tube and connects the head containing the eyepiece to the base of the microscope

what is meant by the term acid fast

that it is not easily decolorized by acids acid fast stain is a differential stiaun used to identify organisms of myobacterium. acid fast can be based on the presence of mycolic acid in their cell wall

Which controls on the microscope affect the amount of light reaching the ocular lens?

the diaphragm and the light intensity adjustment affect the amount of light reaching the ocular lens

stage

the flat platform where you place your slides. Stage clips hold the slides in place and can be moved around by turning two knobs. one of the knobs moves the stage left and right (horizontal movement) the other knob moves the stage up and down (vertical movement)

Which objective focuses closest to the slide when it is in focus

the high power objective lens

Aperture

the hole in the stage that allows the light through for better viewing of the specimen.

the stab tube was inoculated with a needle. Why was this used instead of the inoculating loop?

the inoculating loop is used to transfer specimens. Using a needle will create more of an opportunity for growth to occur along the stab line also showing the bacterias requirements for oxygen

eyepiece lens

the lens at the top of the microscope that you look through

the oil immersion objective is necessary to determine the morphology of prokaryotes

the oil immersion lens is used to increase the resolving power of a microcope by directing more light through the objective lens allowing a closer image to be seen

Condenser lens

the purpose of this lens is to focus the light onto the specimen

parfocal microscope objectives

the specimen being observed remains in focus when magnification is changed.

Why is it desirable that microscope objectives be parfocal?

this is important so that you dont have to refocus the microscope every time you change objective lenses

why is air drying importatn before heat fixing the specimen

to remove water from the stain because it would change the features of the cells if it wasnt

vehicle transmission

transmission by an inanimate reservoir (food, water, air)

vector transmission

transmission of an infectious agent by an insect, arthropod, or animal

vector transmisison

transmitted by the bite of infected arthropod species, such as mosquitoes, ticks, triatomine bugs, sandflies, and blackflies

objective lenses

usually, you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4x (scanning), 10x (low power), 40x (high power) and 100x (oil immersion lenses. when coupled with a 10x (most common eyepiece lens, we get total magnifications of 40x, 100x, 400x and 1000x

The Serratia marcescens cultures were accidentally incubated at 37 degrees Celcius instead of 25 degrees Celcius. You observed growth but the slant and stab cultures were white, not orange-red. does this mean your sample was contaminated?

yes because broth cultures appear to be an off white while slant/stab cultures remain orange/red

Gram stain procedure

1) using aseptic technique make a thin smear of bacteria on the slide. Allow it to air dry and then heat fix it. 2) add a drop or two of crystal violet and allow staining for 1 minute 3) gently wash off the crystal violet using a few drops of DI water 4) add a few drops of iodine solution to the slide and wait for 1 minute 5) wash iodine solution using a few drops of water 6) rinse the slide with decolorizer until no more color washes off. 7) rinse the slide gently with a few drops of water 8) Add a few drops of safranin and allow to stain for 1 minute. 9) rinse the slide gently with a few drops of water 10) blot the slide dry

what is the total magnification if ocular is 20x and objective is 10x

200

A fellow student showed you a gram stained slide where where cells containing cell walls were stained pink. What would you tell her about the staining procedure? Why?

Alcohol removes the lipids in gram negative bacteria which causes the color to wash away allowing the sffranin to be able to counterstain which then turns any gram negative cells pink

List some diseases of upper respiratory system that you learned in lecture that can help you identify the causitive microorganisms using BAP

Bacterial pneumonia upper respiratory infection chronic sinusitus

Why couldn't we examine any anaerobic bacteria that are part of the normal digestive system?

Because the colon consists of mainly strict anaerobes compared to the stomach and small intestine which contains aerobes and facultative anaerobes

Why is food safety important?

GI infections occur due to contaminated food and water. Clean drinking water, washed produce and appropriate storage of food and simple personal hygiene habits such as hand washing help tremendously in reducing the incident of GI infections.

What additional information did the lactose fermentation tubes provide?

Gas production, acid production by fermentation in the tube

What is escherichia coli?

Gram negative rod shaped bacteria living in the gut flora

A fellow student showed you a gram stained slide where cells containing LPS were stained purple. What would ou tell her about the staining procedure? Why?

Gram positive cells are determined by iodine acting as a binder, which shows that gram positive cells are present because of the purple color. If LPS turns purple it means that the stain procedure was done incorrectly by not decolorizing and using safranin.

What does a metallic green sheen indicate on an EMB plate?

Lactose fermentation, usually the bacteria E.coli

blood agar plate

This media is used for the primary isolation of organisms and for subculture of organisms

contact transmission

Transmission of an infectious agent by direct contact of the source or its reservoir with the host.

sexually transmitted

Transmitted by sexual contact

Why is it important to flame neck of the tubes immediately after uncapping and before recapping the tubes?

it prevents airborne contaminents from entering the tubes

coarse adjustment knob

large, round knob on the side of the microscope used for focusing the specimen; it may move either the stage or the upper part of the microscope

Describe the principle, procedure and applications of gram staining

gram staining is used to identify cells based on the differences of the cell wall. While some bacteria may contain a thick layer or peptidoglycan that forms a cell wall, other bacteria have a thin layer of peptidoglycan forming a cell wall sandwhiched between two cell membranes. the outer membrane is rich in lipopolysaccharides (LPS). Gram staining takes advantage of these differences in the cell wall of bacteria. Using two different stains that offer a lot of contrast, bacteria containing a thick cell wall are stained purple. They are called gram positive cells. Bacteria containing a thin cell wall are stained pink and are called gram negative cells. In gram staining, bacteria are first treated with a primary stain, crystal violet. Upon treating with crystal violet all cells are stained purple. Stained bacteria are then treated with iodine. Iodine helps crystalize crystal violet on the cell surface and therefore cells retain crystal violet with higher affinity. After that cells are rinsed with a decolorizing agent, a solution of acetone and alcohol. Decolorizing agent dissolves lipids and peptidoglycan from both gram positive and gram negative cell walls. however the thicker cell wall of the gram positive bacteria will be able to retain some of the cyrstal violet. Lastly bacteria are stained with a counterstain, safranin. Both gram positive and gram negative cells will stain with safranin but the purple crystal violet will override the pink color in gram positive cells. Gram negative cells which are colorless cells after decolorization are stained pink using saftanin.

What method is most commonly used to isolate pure cultures?

he streak plate method. The sample/inoculum is diluted by streaking it across the surface of an agar plate. While streaking in successive areas of the plate, the inoculum is diluted to the point where there is only one bacterial cell deposited every few millimeters on the surface of the agar plate. As these individual bacterial cells divide and produce new bacterial cells, an isolated cluster of cells known as a colony is formed

If you forgot to heat fix the specimen, how will it impact observation of speciment?

heat fixing fixes the bacteria to the slide surface, if this is forgotten the bacteria will smear will be washed off the slide during the staining and decolorizing proxess

A student forgot to use heat during the endospore staining procedure how will it impact the observation?

heat fixing the slide fixes the bacteria to the slide. bacteria can be washed off during staining and decolorization. you woul not see anything under the microscope

If you do not wait 10-20 secontds after flames sterilizing the inoculating instruments before obtaining the sample what might be the consequences?

if it is too hot it will kill the bacteria and it wont grow

fomites

inanimate objects or materials that may contain and harbor bacteria, fungi, or viruses

TSA plates

incubated aerobically and anaerobically (through the use of a GasPak apparatus)

iatrogenic infection

infection transmitted from a health care worker to a patient

Field of view

is the diameter of the circle of light that you see when looking into a microscope. As the power gets greater the field of view gets smaller

the purpose of doing 4 quadrant streaks

is used for the isolation into pure culture of the organisms (mostly bacteria), from mixed population. The inoculum is streaked over the agar surface in such a way that it "thins out" the bacteria.

How is MSA a differential media?

it encourages the growth of a group of certain bacteria while inhibiting the growth of others

Is the gram stain of significant importance in identifying the organisms studied ?

it is significant because it rules out gram negative and rods it does not identify the bacteria of strptococcus