Micro Lab Test 3

Front 1

What kind of culture must be used to run diagnostic tests?

Back 1

pure cultures

Front 2

What are the major types of media

Back 2

General (Complex) Contains necessary nutrients, vitamins, minerals, etc.. Most types of non-fastidious organisms, but not all are able to grow on this type of media. EX: tryptic soy broth, nutrient agar

Enriched-contains added ingredients like blood, serum, vitamins, growth factors etc.. EX: Blood agar

Differential-Contains and indicator designed to cause certain organisms to develope a different appearance from other microbes growing on same medium. EX: MacConkey agar, EMB agar

Selective-Permits growth of certain organisms while preventing growth of others. EX: Mannitol Salt agar, EMB agar

Selective/differential (does both)

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EMB (Eosin Methylene Blue) Agar

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Purplish Color

Selective for gram negatives

Differential for lactose fermenters and fecal

coliforms

+ result=dark purple colonies. Produces the enzyme b-galactosidase to ferment lactose to an acidic by-product. If pink colonies show it means it's a slow lactose fermenter.

- result=colorless colonies. Appear same color as EMB

Coliforms: green metallic sheen on colonies. Denotes vigorous lactose fermentation to an acidic by product.

Result must be read in 24 hrs.

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MacConkey Agar

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Selective for gram negative

Differential for lactose fermenters.

+ lactose fermenation=pink-lavendar colonies. Produces the enzyme B-galactosidase to ferment lactose to acidic by products.

- lactose fermenation: colorless colonies.

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Starch agar

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Differential for bacteria that can hydrolyze starch; like bacillus, corynebacterium and clostridium.

+starch hydrolysis: clear zone (often golden color halo) around bacterial growth AFTER adding iodine. Produces alpha-amylase oligo 1, 6-glucosidase to break down starch into smaller sugar subunits.

-starch hydrolysis: dark blue or brown agar (no clear zone) after adding iodine.

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MSA (mannitol salt agar)

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Selective for staphylococcus species (7.5% salt)

Differential for mannitol fermenters. Phenol red dye detects acid production by turning yellow.

+ mannitol fermentation- yellow agar. produces mannitol dehydrogenase to break down mannitol to acidic by products.

- mannitol fermen. color of origional red agar.

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Blood Agar

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Tryptic Soy agar enriched with 5 or 10% whole sheep blood. It is widely used in the culture of medical specimens and for many types of fastidious bacteria.

Differential for hemolysis reactions.

Gamma Hemolysis: no change in agar appearance; absence of hemolysis

Alpha hemolysis: greenish color to agar; partial destruction of RBC's.

Beta hemolysis: Clear zone around colonies; total destruction of RBC's.

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Columbia CNA (Colistin-nalidixic acid) with 5% sheep blood

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Selective for growth of gram positives, particularly staphylococci, streptococci and enterococci. Inhibits the growth of gram negatives b/c of the presence of the antibiotic colistin and nalidixic acid.

Enriched with the addition of blood

Differential for hemolytic reactions

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Hektoen Enteric (HE) agar

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differential for lactose fermentation and hydrogen sulfide production

Selective for mainly salmonella, shigella; E. coli can also grow on it.

+ Lactose fermen=yellow to salmon-pink colonies. fermentation of lactose sugar produces acidic by products.

-lactose fermen=blue-green colonies.

+hydrogen sulfide-black colonies. (Reduces Sulfur to H2S which reacts with ferric ammonium citrate in the medium to create a black precipitate.

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Triple Sugar Iron (TSI) Agar

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Differential for glucose, sucrose and/or lactose fermentation; also hydrogen sulfide production. Helps to distinguish gram- enteric bacilli in the family Enterobacteriaceae.

Contains iron.

-fermentation=red slant, red butt

+ fermentation of only glucose=red slant, yellow butt (glucose broken down to acidic by product)

+ fermentation of glucose, lactose and or sucrose=yellow slant, yellow butt (2/3 sugars broken down to acidic by products)

+hydrogen sulfide (H2s) blackened media; sulfur reduced to H2S (H2s reacts with ferrous sulfate in medium to form black precipitate)

+ gas; lifting of agar or appearance of cracks in agar

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Phenol Red Carbohydrate Fermentation Broth

Back 11

Differential for carbohydrate fermentation using pH indicator (phenol red)

Can be designed to test any one specific sugar. Used to identify bacteria in general, especially useful in distinguishing gram neg. enteric bacilli but may be used for other organisms too.

+ fermentation: Yellow broth (the specific carbohydrate used) is broken down into acidic by-products.

- fermentation: original red broth

+ gas production: sometimes gas is produced as a result of fermentation and is seen as bubblescollected in the Durham tube. (upside down tube)

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Citrate agar slant

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Differential for citrate utilization. If microbe can use citrate as it's only available source for carbon. Useful in identifying Gram negative enteric bacilli in the family Enterobacteriaceae

+ citrate= any amounts of blue. Organism produces citrate – permease to transport citrate (only source of carbon) into its cells to be utilized.

- citrate=green

* A heavy amount of culture is needed

Front 13

Urea Broth

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Differential for urease production. Useful in distinguishing Proteus (urease +) from salmonella or shigella (urease -).

Urease is an enzyme that breaks down urea to ammonia

Phenol red (pH indicator) changes to bright pink at alkaline pH

+ urease=dark pink (urease hydrolyzed urea to ammonia)

- urea=original salmon (orangish) or yellow color.

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Phenylalanine Agar

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Differential for phenylalanine deaminase production. Helps to distinguish between gram negative enteric bacilli.

+ phenylalanine deaminase: green, when iron III chloride added. Organism has broken down phenylalanine to phenylpyruvic acid using phenylalanine deaminase.

- phenylalanine deaminase: no color change (except for added chemical)

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SIM Medium (semisolid)

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Differential for hydrogen sulfide production; motility and indole production. Especially useful in identifying gram negative enteric bacilli in the Family Enterobacteriaceae.

3 tests are run in ONE tube of medium--indole production, hydrogen sulfide (H2S) production and motility

a. hydrogen sulfide (H2S)

+ H2S: blackened medium; H2S reacts with ferrous ammonium sulfate to produce the balck precipitate, ferric sulfide

- H2S: :No color change

b. Motility

1. Indicates if organism possesses flagella for movement.

+ motility: growth diffusing away from stab line indicates flagella are present.

- motility: growth only along stab line

c. Indole production

+pink or red ring when Kovac's reagent is added; break down of amino acid, tryptophan by enzyme, tryptophanase to indole.

- indole; no color chage when Kovac's added.

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Motility/ GI medium/TCC (MOT)

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Can be used as an alternate to SIM due to SIM's difficult interpretation.

Differential for determining whether the organism has flagella as a means of movement. It contains an indicator called triphenyltrazolium chloride (TCC) which turns red in the presence of bacteria. This color traces the growth of the bacteria in the medium.

+ motility: medium displays a diffuse pattern (turbidity) of red color away from stab line or throughout the medium indicates organism possesses flagella.

- motiltiy: red color only along the line of inoculation and no turbidity in the rest of the medium.

Front 17

optichin disk susceptibility Test

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paper disk impregnated with the scant amount of the antibiotic optichin. It is used to presumptively identify Strep pneumoniae which is sensitive to it, from other species of streptococci.

The organism to be tested is "painted" on a Blood agar plate and an optichin disk is placed in the middle of the "Lawn" It's then incubared at 35c in a candle jar for 24 hrs.

+ result: (presumptive for S. pneumoniae)14+ mm zone of inhibition around a 6 mm optichin disk.

- result: smaller inhibition zones indicate further testing required. Likely organism is not S. pneumoniae.

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Coagulase Test

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Distinguishes Staph. aureus from other gram positive cocci

The enzyme, coagulase, causes blood plasma to clot

+ coagulase: clotting of plasma (solid) indicates coagulate produced

- coagulase: no clotting (liquid)

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Oxidase Test

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Cytochorme c oxidase is an enzyme oxidizes tetramethyl-p-phenylenediamine (colorless) into a colored by-product. It is used to distinguish Pseudomonasand Neisseria from other organisms.

+ oxidase: purple or black; Cytochrome c Oxidase produced which oxidizes tetramethyl-p-phenylenediamine into a colored by product.

-oxidase: no color change.

Front 20

Catalase Test

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Helpful in distinguishing Staphylococcus from Streptococcus.

Catalse breaks down hydrogen peroxide (H2O2) to water and oxygen gas.

+ catalase: any bubbling; enzyme catalase produced by organism which broke down H2O2 to H2O and O2

-Catalase: no bubbling

Front 21

Chocolate agar

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Cooked blood agar. Will grow a wide range of organisms. Contains X & V growth factors which will allow the growth of Neisseria and Hemophilus.

Used for respiratory, eye, ear, nose and throat specimens.

NOT used for stool or wound specimens.

Front 22

Does refrigeration or freezing stop all bacterial growth?

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NO. refrigeration slows it down and freezing halts it but will resume once thawed.

Front 23

Staphylococcal food poisoning

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Causative agent: S. areus.

Causes food intoxication

Staphylococcal food poisoning is a gastrointestinal illness. It is caused by eating foods contaminated with toxins produced by Staphylococcus aureus . The most common way for food to be contaminated with Staphylococcus is through contact with food workers who carry the bacteria or through contaminated milk and cheeses.

Front 24

Food infection

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An overgrowth of bacteria that establishes themselves in GI tract

Front 25

Food intoxication

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Bacteria release toxins that make you sick

Front 26

Fecal coliform

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facultatively anaerobic, rod-shaped, gram-negative, non-sporulating bacterium. This bacteria generally originate in the intestines of warm-blooded animals.

Front 27

Salmonellosis

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Caused by salmonella; food infection.

To avoid: Cook poultry, ground beef, and eggs thoroughly. Do not eat or drink foods containing raw eggs, or raw (unpasteurized) milk.

Front 28

Shigellosis

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Caused by shigellosa; food infection
A safe water supply is important for the control. and is probably the single most important factor in areas with substandard sanitation facilities
Transmitted by the fecal-oral route, most infections are transmitted from person to person. Where persons who are infected may be present, the risk of transmission and infection increases with poor hand hygiene, ingestion of contaminated food or water, inadequate sanitation and toileting, overcrowding, and sexual contact.
Because of its quite common person-to-person spread,it has long been associated with outbreaks in daycare centers, nursing homes, institutional settings (like prison), and cruise ships.

Front 29

E.coli; gastroenteritis (all types)

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Caused by E. coli Food infection.
Wash utensils. Use hot soapy water on knives, countertops and cutting boards before and after they come into contact with fresh produce or raw meat.Keep raw foods separate. This includes using separate cutting boards for raw meat and foods, such as vegetables and fruits. ... Wash your hands.

Front 30

botulism

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caused by colostridium; food intoxication

commonly found in soil, on raw fruits and vegetables, on meat and fish, and on many other foods and surfaces.Their spores are tough, and cannot be killed with boiling water or heat without including canning pressures. Bacteria (the bacteria that grow out of germinated spores) can multiply quickly in a moist, oxygen-free environment and create a very powerful poison. One teaspoonful is enough to kill 100,000 people.Improper home canning creates the perfect environment to grow bacteria. bacteria.Because food contaminated by this bacteria may look and smell normal, you cannot tell by looking at the food whether it is poisoned by this bacteria.

Front 31

What are the different types of agar plates

Back 31

EMB

MacConkey Agar

MSA

Starch Agar

Blood Agar

Columbia CNA

HE Agar

Front 32

What are the different broth mediums

Back 32

TSI Agar

Phenol Red

Citrate Slant

Urea broth

PA

SIM

MOT/TCC

Front 33

What enzyme is useful in detecting is E. coli?

Back 33

beta-D-glucuronidase and tryptophanase (indole formation)

Front 34

what are the characteristics and diagnostic media results for E. coli?

Back 34

Gram negative rod shaped bacillus usually found in the gut of warm blooded mammals.

MacConkey Agar test= + for lactose

SIM= + for indole

MOT/TCC= +

Citrate Agar= Negative

Urea broth= Negative

PA= negative for deaminase production

H2S=Negative

Front 35

what are the characteristics and diagnostic media results for Salmonella

Back 35

Gram negative Rod shaped aerobic bacteria possessing flagella.

MacConkey Agar= Neg. lactose

MOT/TCC=Pos.

Citrate Agar=Positive

Urea=Neg

PA=Neg

H2S=Positive

Front 36

what are the characteristics and diagnostic media results for Proteus mirabilis

Back 36

Gram-negative, facultatively anaerobic, rod-shaped bacterium.

MacConkey Agar= Neg. lactose

MOT/TCC=Pos.

Citrate Agar=Negative

Urea=Pos.

PA=Pos.

H2S=Positive

Front 37

what are the characteristics and diagnostic media results for bacillis subtilis

Back 37

cells are rod-shaped, has spores, Gram-positive bacteria that are naturally found in soil and vegetation

Catalase: Positive

MOT/TCC: Positive

Starch Agar: Positive

Phenol Red: weakly positive

Oxidase: +/-

Citrate: positive

Front 38

what are the characteristics and diagnostic media results for Staphylococcus areus

Back 38

gram + cocci in clusters, non motile, non spore forming, facultatively anaerobic

Catalase: Positive

Phenol Red: Positive glucose ferment.

Coagulase Plasma: Positive

Mannitol Salt Agar: Positive mannitol ferment.

Front 39

what are the characteristics and diagnostic media results for Staph. epidermis

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Gram + cocci, non motile, often appear in pairs or tetrads. May also appear as single cells or in small clusters. Facultatively anaerobic

Catalase: Positive

Phenol red: Positive

Coagulase: Neg.

Mannitol Salt agar: Neg.

Front 40

what is an epidemiologist

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scientist who fights infectious diseases. and study the causative agents of disease. ie; virus', bacteria, fungi, protozoa.

Front 41

Contact transmission

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the physical meeting of a source and a new host, often through person to person contact. ie: touching, shaking hands, kissing, direct contact w/body lesions, intimate sexual contact and mother to infant via breast milk.

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Indirect contact

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transmission through fomites. Pseudomonas are commonly transmitted this way.

Front 43

droplet spread

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when a pathogen is carried on a particle larger than 5 micrometers. ie; sneezing, coughing

Front 44

Which organism in most frequently found in human infections?

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Staphylococci. Most common infections by S. aureus are skin infection but more serious illnesses such as bacteremia, endocarditis and meningitis may be observed in the hospital environment.

Front 45

ASI Staphslide latex test

Back 45

aggultination test that detects clumping factor (coagulase) and protein A by plasma coated latex particles.

Tests for Staph. aureus

Front 46

EnteroPluri Test Rapid ID

Back 46

Test offers 15 biochemical tests in 12 compartments within a self contained plastic tube. tube contains its own sterile inoculating wire. Bacterial specimen must be a pure culture of a gram negative bacillus that is oxidase negative.

all negatives read as "0" and positives are given a number.

Front 47

ELISA test

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a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions.

Indirect: looks for antibodies of patient

Direct: looks to antigen

Front 48

How much bacteria is normally found in a gram of hamburger

Back 48

1 mil/gram. That's why it has such a short shelf life.

Front 49

What were the results of the RT and Refrig. beef culture on the EMB plates?

Back 49

Growth exhibiting a metallic shiny green sheen=coliforms
Dark purple colonies=lactose fermentation
Green sheen + purple colony = probably E. coli.

RT beef had more growth on it. Showed presence of salmonella.

Front 50

What were the results of RT and refrig beef on HE Agar

Back 50

Blue-green colonies = negative lactose fermentation and most likely are salmonella or shigella. Colonies which are yellow to salmon pink are lactose fermenters and may be E. coli or other coliforms. Black/grey color= H2S (hydrogen sulfide)

Front 51

What were the numbers of bacteria per gram for chicken

Back 51

Room temp= 1: 100/million per gram

Refrig= 1: 10 million per gram

Front 52

Urine is sterile when it leaves the kidneys? T or F

Back 52

True. As it passes through the urethra it picks up bacteria that normally inhabit the surface tissues of the lower end of the urethra near the external opening. Bacteria in urine doesn't = infection. Only if there's a large # of organisms present.

Front 53

What is the most common bacterial offender for bladder and UTi's

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E. coli. Usually due to improper anal hygiene. Women are also more succeptible to get it due to the smaller urethra.

Other pathogens to cause infection are:

Proteus, pseudomonas aeruginosa, enterococci, candida albicans, Staph. aureus, Staph saprophyticus (becoming more common especially in young sexually active females) myobacterium tuberculosis, salmonella and shgella and Klebsiella.

Front 54

What are some routes of entry for Bladder infections

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Contamination by Coliforms- E.coli

Catheterization-especially pseudomonas, proteus and staphylococcus.

Direct contact- with infected persons. Organisms can get on fingers and then person can touch genitalia, sexual contact as well.

Tight pants

synthetic underwear-cotton is better

multiple layers of clothing

improper hygiene

bubble bath-soap dries out urethral opening

sex

Front 55

How can bacteria gain access to the kidneys and cause infection

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1. Hematogenous (via blood) Bacteria from other pars of the body can gain access to the kidneys via the blood and set up infection

2. Lymphatic System-they can be carried to the kidneys through lymph system

3. ureter-they can ascend the ureters up to the kidneys to establish infections

Front 56

how many bacteria in urine is considered an infection

Back 56

A culture with excess of 10,000 may indicate the beginning of an infection.

100,000 or more organisms per ml indicate a urinary tract infection.

Normal urine contains 100-1,000 colonies of bacteria per ml of urine. Usually from normal skin flora around urethral opening.

any bacteria present in a catheter bag should be considered an infection as the bag and the urine it collects should be sterile.

Front 57

What is a clean catch

Back 57

properly cleaning the genitalia with moist wipes and providing a "midstream catch" of urine to be cultured to check for infection

Front 58

What could cause false results when culturing a urine specimen

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normal flora contaminants in urine that may overrun pathogen

Overgrowth of normal flora due to delay in culturing or specimen not refrigerated

Delay may cause death of fastidious pathogens.

Front 59

How is the total number of bacteria per ml of urine found

Back 59

By the number of colonies on a streak plate X 100 (diluation) X 100 (calibrated loop)

Front 60

What is a calibrated gram stain

Back 60

fast method of determining the gram reaction and morphology, and also the number of cells per milliliter of urine.

A slide with a 1-cm square grid etched in it is smeared with a designated amount of the urine specimen.

A large number of cells appearing on the slide calculated by using the grid, indicates a possible infection.

This is usually used in time sensitive situations like day of surgery . It is not very accurate.

Front 61

Which mediums should be used for human specimen cultures

Back 61

1. Blood Agar-grows many types of human pathogens.

2. MacConkey or EMB (selective for gram -)

3. CNA blood agar (selective for gram+)

4. Hektoen enteric agar (select. for gram - enteric bacilli)

5.Thioglycolate Medium (promotes growth of very fastidious aerobes and anaerobes.

6.Lowenstein-Jensen medium-should be used if mycobacterium tuberculosis is suspected.

Front 62

How does an antibiotic sensitivity test benefit a patient that comes in w/an infection

Back 62

so that treatment can be started before infection has time to worsen or spread. Usually a broad spectrum antibiotic is prescribed until the results of the tests are known and the drug can be changed if necessary.

Front 63

Gonorrha

Back 63

Caused by Neisseria gonorrhea. also known as gonococci (plural), or gonococcus (singular), is a species of Gram-negative coffee bean-shaped diplococci bacteri. Spread through sexual contact.

Front 64

Syphilis

Back 64

caused by Treponema pallidum is a Gram-negative bacteria which is spiral in shape. It is an obligate internal parasite. Has to be gram stained with due to it's high lipid content. Spread from person to person by direct contact with a sore, known as a chancre. Chancres occur mainly on the external genitals, vagina, anus, or in the rectum. Chancres also can occur on the lips and in the mouth. Transmission occurs during vaginal, anal, or oral sex.

Front 65

what basic principles of antibody mediated immunity are utilized in an ELISA assay

Back 65

If antibodies are present in patient

Front 66

How does an ELISA indirectly detect infection by a disease causing agent

Back 66

determines antibody

Front 67

What is the function of the secondary antibody and chromogen in an ELISA

Back 67

Secondary antibody is attached to an enzyme. when chromogen is added if a patients has the antibodies the enzyme that is linked to the secondary antibody facilitates a chemical reaction that changes the color of the chromogen. A color changes indicates patient has antibodies to illness.

Front 68

Lyme Disease

Back 68

spirochete bacterium-borrelia bergdorferi

Transmitted to people through the bite of infected ticks, such as deer and black legged. Can't be xfered from person to person. They require meals of blood obtained from warm blooded animals. After it bites victim it transf. the infectious bacteria after 36-48 hrs of attachment. Symptoms include: bull's eye rash surrounding bite, flu like symptoms, headache, chills, muscle aches and fatigue. If untreated neurological difficulties and respiratory and GI problems can happen.

Front 69

Pricipitin Ring Test

Back 69

Precipitation reactions are based on the interaction of antibodies and antigens. They are based on two soluble reactants that come together to make one insoluble product, the precipitate . These reactions depend on the formation of lattices (cross-links) when antigen and antibody exist in optimal proportions.