MBIO Lab Exam 1

Urine MUST be culture within _________ hours after collection or if refrigerated within _________ hours.

1-2 hours 24 hours

What is soil composed of?

1. Inorganic material (40%) 2. Organic matter (5%) 3. Air & H2O (50%) 4. Microorganisms and macroorganisms (5%)

Why is colony morphology important?

1. It can give you an idea of how many different bacterial species are in a sample. 2. It provides you with the first clue as to the identity of the bacterial species in a sample.

Studies examining hand hygiene compliance rates in healthcare works found only ___________ compliance

50-63%

__________ consist of two components joined by a chemical link: Chromophore which gives the stain its _________. Auxochrome which is the _________ portion of the stain.

A chromagen Color Charged

To make media __________ you have to have a "thickener." In microbiology we use __________ which comes from seaweed.

A solid Agar

__________ are a class of bacteria that also produce antibiotics.

Actinobacteria

Soaps and waterless hand cleaners are _________________ in that they ___________ the concentration of microbes on skin

Antiseptics Reduce

The three types of organisms we find in our world are: Free-living Microorganisms... which __________ associated with any specific organism and are __________ pathogenic (disease causing). Pathogenic Microorganisms... which __________ disease. Opportunistic Pathogens ... which __________ inhabit our bodies ___________ causing disease, but if introduced to a new area or if immune system is suppressed can cause disease.

Are not Non-pathogenic (do not causes diseases) Cause Normally Without

When carrying the microscope always use two (2) hands; one on the _________ and one under the __________ of the microscope

Arm Base

We "move" bacteria around in the lab using the methods of __________ __________ and __________ for maintaining and testing.

Aseptic transfer and innoculation

What are the three kinds of tape you are likely to use in the lab and why? ♥

Autoclave indicator tape Masking tape- to keep stacks of plates together

How can you tell whether or not something has been autoclaved and is sterile?

Autoclave indicator tape Stripes on the tape darken during the steam sterilization process

STERILITY is the single most important thing about making media. The most effective way of sterilizing media is by __________.

Autoclaving

Why would UTIs be so much more common in the hospital environment?

Because catheters are used

A _________ is a complex association that arises from a mixture of microorganisms growing together on a surface. What are a few examples of biofilms?

Biofilm •the scum on the surface of a stagnant pond •the scum on the inside of water/sewage pipes •the scum on the hull of a boat •the scum on diseased fruits and vegetables •the dental plaque on teeth

What goes in the pails on the discard cart at the back of the lab?

Biohazards such as petri dishes

Slow-Exhaust which is used for "__________" so they don't bubble over.

Bubble over

Diagnosis of a UTI is based on the number of __________ in the sample. Determining CFU/ml by serial dilution and spread plates is _________ practical in a diagnostic lab. So we use an alternate method.

CFU/ml Not

However, 1 bacterial _________ can form a colony. (A visible colony is hundreds of thousands of cells that started from one bacterial cell that has _________ into a group of _________ cells.)

Cell Divided Identical

Stains are classified positive or negative based on the charge carried by the auxochrome. It is this __________ that determines whether a stain "sticks" or is "repelled" from the bacterium

Charge from the chromagen

There needs to be hundreds of thousands of bacterial cells in order to form a ________ which is visible to the naked eye

Colony

In lab we use a __________ __________ meaning it has two (2) sets of lenses. Ocular Lenses located on the _________. _________ Lenses are on the nose piece.

Compound microscope Eyepiece Objective

If the dilutions and spread plates are done correctly the colonies on each plate should _________ in number as the dilution __________

Decrease Increases

The number of microbes _________ as you go deeper into the soil layers.

Decreases

A __________ media is one in which we know exactly all of the ingredients and amounts. Since there are generally many ingredients that go into this type of media, it is a pain to make!

Defined

Streaking for isolation is a __________ technique. Meaning you start off with a _________ concentration of mixed/non mixed bacteria and as you streak the concentration of the bacteria _________. Finally the bacteria are at such low concentration that individual cells on the agar surface can form a __________.

Dilution High Reduces Colony

Bacterial pathogens are bacterial species that can cause _________ in plants/animals.

Disease

If you want to reduce the volume of the dilution you are making you __________ all the volumes used in the dilution by the same number. To __________ the volume of the dilution you would multiply all the volumes by the same number. ♥

Divide

Fast-Exhaust which is used for "__________" things like glass, instruments, swabs, etc.

Dry

The bacteria _________ _________ is most commonly associated with UTIs

E. Coli

Steam can be released from the chamber of an autoclave in 1 of 2 ways

Fast Exhaust - for "dry" things like glass bottles, flasks or medical equipment Slow Exhaust - for liquids so they don't "bubble over" (you can lose a lot of media if it does)

What are a few of the things to be considered when talking about morphology?

Form Elevation Margin Pigmentation Texture Growth patterns

The purpose of lab is to provide a __________ - __________ in understanding the concepts of microbiology. Everything you see and do in lab are cornerstones of microbiology practiced in the "__________ __________."

Hands-on Real world

Positive stains requires the smear to be __________ fixed before staining. What does this heating do to the cell? What is a disadvantage?

Heat •Requires a bacterial smear to be heat fixed prior to staining •Heat fixation kills the bacteria and secures the dead organisms to the slide; however, it can somewhat distort the bacterial cell shape

Understanding ubiquity is important for everyone ( health-care workers and __________) to understand since _____ out of _____ patients will acquire an infection in the hospital which could be life threatening.

Hospital visitors They need to understand the importance of hygiene since microorganisms lurk all around us

Things coming out of the autoclave are extremely __________ and require you wear protection so you are not burned!

Hot

When viewing a specimen under the 100x objective you must use __________ __________. This is necessary so the light going up through the specimen remains _________. Without the oil light is refracted away from the objective and would be blurred.

Immersion oil Unrefractive

Why is it important in a healthcare setting to know how to make dilutions?

In a hospital setting you may be required to dilute drugs prior to administration

The fire extinguisher, eye wash station, and chemical station are all located in the hall while the first aid kit is located

In the front bench cabinet

Contaminated media plates, plastic tubes, swabs, etc. go in the __________ __________ __________.

In the pails on the discard cart at the back of the lab

Chemoautotrophs oxidize __________ compounds to yield energy and reduce CO2.

Inorganic compounds

In all fields of microbiology it is absolutely critical to be able to __________ bacteria. The goal of isolation is to obtain a __________ culture from a __________ culture containing two or more species.

Isolate Pure Mixed

The goal of quadrant streaking is to get __________ colonies.

Isolated

Use only _________ _________ to remove oil from lenses. Never, ever use paper towels, Kimwipes or ________ Paper since they will scratch the lens.

Lens paper Bibulous

Biofilms are medically important and can lead to _________ _________ infections. List a few "indwelling devices" that are susceptible to biofilm formation.

Life threatening •contact lens •urinary catheters •mechanical heart valves •pacemakers •prosthetic joints

To enlarge the image of the specimen, light is emitted from the _________ _________, then it enters the CONDENSOR which _________ the light beam that strikes the specimen. The light that passes through the specimen enters the __________ lens that enlarges the image and passes through the _________ lens that makes the final enlargement

Light source Narrows Objective Ocular

Two techniques that are important in determining/quantifying bacteria:

Making dilutions and making spread plates

When using an autoclave you also need to remember the more you put into it the __________ time it takes to reach sterilization temperatures.

More

Bacteria have a __________ charge on their surface. The old rule about opposites attract applies here. This would mean that a _________ stain would stick to (and color) the bacteria while leaving the background white. A __________ stain would be repelled by the bacteria leaving the bacteria colorless and the __________ stained.

Negative Positive Negative Background

1 colony does _________ equal 1 bacterial cell. (One bacterium is ________ visible on a plate.)

Not Not

An undefined media is one in which the exact ingredients and amounts are __________ known.

Not •Example: beef extract used in many different media is the concentrated broth that comes from cooking beef •Commercially available and easy to make (usually only a couple of ingredients)

Once the __________ of colonies at a __________ dilution is known then it is possible to calculate the number of __________ _________ _________ (CFU) in 1ml of sample. ♥

Number Specific Colony forming units

Just like us bacteria need __________ to survive. These can be found in ___________ (liquid) and __________ (solid) forms. There are many different types of media since some organisms are very "__________ / selective." If we can't grow the microorganisms we can't study them!

Nutrients Broth Agar Picky

What is the rule for using plates in your CFU/ml calculations?

Only use plates with between 20/30 to 200/300 colonies for calculations

Decomposers break down __________ biomass/compounds.

Organic

Using the plate counting rule ( 20/30 - 200/300) we can determine the __________ __________ __________ (OCD). OCD = (# of colonies)/(loop volume) CFU/ml

Original cell density

Bacterial mutualists form _______ with plants

Partnerships

The purpose of sub-culturing is to obtain a _________ culture of a bacterial species. Pure cultures contain only one type of bacteria.

Pure

The bacteria within a biofilm are generally more ________ to disinfectants and ________. Once a biofilm forms it is ________ _________ to remove from the surface of a device.

Resistant Antibiotics Extremely difficult

Species of bacteria that require a plant host (for nutrients and protection) and in return fix nitrogen (N2 gas to ammonium) that can be used by the plant

Rhizobium

When you are making dilutions there are 3 very important points to remember: Always add the __________ to the saline/broth Always __________ each dilution thoroughly after adding the sample. Always, Always change _________ tips after __________ transfer.

Sample Mix Pipet

What is the slow exhaust analogy?

Shake up a carbonated drink in a bottle and the contents will be under extreme pressure. If you quickly open the cap the contents will shoot out every where as the pressure is released (fast exhaust). If you very slowly open the cap releasing the pressure a little bit at a time then the contents will remain in the bottle when the cap is finally removed (slow exhaust)

A __________ is a solution in which a dye or a chromogen has been added.

Stain

Steps to making a spread plate

Step 1 •label each plate according to the tube the sample will be taken from (e.g., Tube #4 10-4) •put 0.1 ml of the bacterial dilution onto the MIDDLE of the agar plate Step 2 •put a METAL HOCKEY STICK into a beaker or jar of ALCOHOL •Remove the stick and let any EXCESS ALCOHOL on the stick drip back into the beaker or jar Step 3 •pass the metal hockey stick over a LIT Bunsen burner so that the ALCOHOL IGNITES and kills any bacteria on the stick Step 4 •COOL the hockey stick by touching it to the agar away from the bacterial solution •SPREAD the sample over the ENTIRE surface of the plate with the glass hockey stick being careful not to rip or gouge the agar STEP 5 •once the bacterial solution has been spread over the surface of the plate allow the solution to be ABSORBED into the agar •after the solution has been absorbed turn the plate over and incubate overnight

The single most important thing when making media is

Sterility

Disease Suppressors are bacteria that _________ the occurrence of plant disease by secreting anti-fungal or insecticidal compounds

Suppress

Don't be afraid or embarrassed to tell your __________ of mistakes and accidents. It is better to be ___________ than sorry!

TA Safe

3 parameters critical for obtaining sterility: Also, why are all 2 parameters necessary?

Temperature Time And pressure (from steam) It's one of the only effective ways to KILL SPORES

Why is it important to learn how to determine/quantify the number of bacterial colonies in a sample?

The Importance of Colony Morphology 1. It can give you an idea of how many different bacterial species are in a sample. 2. It provides you with the first clue as to the identity of the bacterial species in a sample.

What roles do microbes play in the soil?

The carbon cycle The nitrogen cycle The sulfur cycle It is a key player in the soil food web

Non-contaminated paper and towels soaked with Amphyl go in the __________ _________.

The empty crock at the end of the lab

When sterilizing your loop or needle in a flame which cone do you pass it through? Why

The inner flame in order to heat it sufficiently to turn the loop a uniform orange color

How do you know you have heated the loop or needle sufficiently?

The loop will turn an orange color

Why do bacteria need to be stained? Cytoplasm is __________. Stains/Dyes provide _________.

Transparent Contrast You would not be able to see the bacteria if it wasn't stained

Urinary Tract Infections ( _________) are the 2nd most common type of infection. It is the number one type of __________ acquired infection.

UTIs Hospital - most likely due to catheterization (insertion of a tube into the bladder)

What is a simple definition of ubiquity?

Ubiquity means existing or being everywhere

What goes in the broken glass box at the back of the lab?

Uncontaminated broken glass such as stained microscope slides

Media is considered either _______ or ________

Undefined Defined

First a __________ loop is used to transfer a __________ volume of sample to a Blood Agar Plate (BAP). This special plate contains sheep's blood and is __________ rich so many different bacterial species can grow on it. Then the sample is spread out over the entire surface of the plate using a _________ streaking method called a __________ - _________ streak.

Volumetric Nutrient Specific Semi-quantitative

Contaminated slides that have not been heat-fixed, used pipettes, etc. go in the __________ __________ on the lab bench.

Waste bin thing

What is Actinobacteria?

class of bacteria that resemble fungi (branched networks of hyphae) and produce numerous enzymes that help break down organic plant material Also produces antibiotics

Why do you streak environmental samples (zigzag) and clinical samples (quadrant/urine) differently?

• Unlike clinical samples, the amount of bacteria in an environmental sample is likely to be much lower • If there is a low density of bacteria to begin with, quadrant streaking for isolation would not be effective since the bacteria would likely all appear in the 1st quadrant and not be well separated • Quadrant streaking for isolation is difficult away from a lab setting since there is generally not a flame source available.

What is the primary reason for using aseptic techniques to maintaining sterility of our cultures/sample?

•Contamination of a culture with additional bacterial species can confound results used in the identification of pathogenic bacterium and could lead to the wrong diagnosis of an infectious disease We want to prevent the media from getting contaminated

Why is obtaining a pure culture so important?

•If you have a sample with more than one bacterial species any test you perform on that culture will be unreliable because the test results may be from any one of the organisms in that culture Example: A lab technician receives a sample taken from a patient's infected wound. That sample contains many different bacterial species including the one causing the infection. The doctor needs to know what that species is before he/she can treat the patient. The technician needs to separate out all the bacterial species in that specimen in order to identify the one species causing the infection.

Why should you keep your tubes in the test-tube rack and other work items in a convenient place?

•Make sure your culture tubes are in the test tube rack (tubes resting on the bench are likely to leak, roll off the bench and break or both) and that everything you need for your work is in a convenient place (do you really want to have to lean/reach over a lit Bunsen burner to get something?)

Negative stains should _______ be heat fixed. The advantage to not heating the smear?

•Should NOT heat fix the slide •Causes minimal cell distortion so can better discern size and cell arrangements/patterns

What are 2 important things to consider with a standard autoclave?

•The amount of time required for sterilization (the more you put into the autoclave the more time is required for sterilization) •How fast the steam is released from the autoclave