MCB 2010C Microbiology Lab Practical Midterm Prof. Curtis (Valencia)

Trypticase Soy Broth (TSB) organisms analyzing temperature

-e. coli - growth at 37 - true mesophiles -Serratoa marcescens - growth at 37 - True mesophiles -Bacillus stearothermophilus - 60 degree C - True Thermophile (not a concern - to cold for human host) -Pseudomonas fluorescens - 5 degree C - psychrotrophs

MacConkey Agar (MAC) - (favors gram (-))

1) Enterobacter aerogenes/pink/gram negative/fermentation 2) Escherichia coli / pink / gram negative /fermentation 3) Enterococcus faecalis / No growth / gram positive 4) Salmonella typhimurium - growth, no color change / gram negative / no fermentation Fermentation will produce acid. When they ferment sugar they will make acid as a byproduct.

Eosin Methylene Blue Agar (EMB) Organisms

1) Escherichia coli - black/dark purple )glows green) - Yes, lactose fermentation - gram negative 2) Salmonella typhimurium - purple / Yes, lactose fermentation - gram negative 3) Enterococcus faecalis - almost clear / Yes, lactose fermentation - gram negative 4) Enterobacter aerogenes - Pink / Yes, lactose fermentation - gram negative

Endospore Stain Procedure

1) Prepare smear with Clostridium botulinum , same technique as simple stain procedure. 2) Air dry and heat fix smear and place on staining rack 3) Add primary stain (Malachite green) for 60 sec in the presence of heat. Heat acts as the mordant. 4) Gently heat the slide with stain on it by passing bunsen burner under slide until slide steams (heat is mordant) 5) Decolorize for 15 Sec with water. 7) Add the counterstain Cover smear with Safranin for 1 minute. 8) Pour off excess Safranin and use water bottle to rinse any left over safranin from slide 9) Gently blot slide using bibulous paper, then observe slide with immersion oil

Acid-Fast Stain Procedure

1) Smear a small amount of Myobacterium onto a dry slide (break up any clumps of cells with loop) and spread them out, after they are spread add water the the smear. Transfer a small amount of Staphylococcus aureus onto smear. 2) Air dry AND Heat fix smear 3) Cover entire slide with Carbol Fuschin 4) Gently heat slide by passing the flame from bunsen burner under slide until it begins to steam. Drives carbon fuschin into the walls of acid- fast bacteria 5) Steam slide for a total of 5 minutes. Replenish carbon fuschin as it begins to evaporate 6) Pour off excess Carbol Fuschin and let slide COOL 7) Once slide is cool, rinse with water 8) Decolorize the smear with Acid-Alcohol mixture, holding slide at 45 degree angle and rinsing until mixture runs clear. 9) Rinse remaining acid alcohol off with water 10) Counter stain smear with Methylene Blue for 1 minute, do not cover the whole slide 11) Gently rinse smear with water and blot dry with bibulous paper and look at with immersion oil lense

Why is it important to use a smear preperation before doing a simple or differential staining technique?

1) it adheres the cells to the glass slide so you can view the specimen properly 2) for safety purposes, it is important to kill the living microbes on the slide.

heat-fixing your slide when making a smear prep

1) kills the microbes making the slide safer to handle and making the cells walls more porous to facilitate taking up the basic/cationic dyes. 2) it melts the surface sugars (such as peptidoglycan and lipopolysaccharide) causing the cells to stick to the slide.

At what position should you hold the agar plate when doing your inoculations to minimize the likelihood of contaimination

90 degree angle

When do you blot your slides dry using the booklet of bibulous paper during the staining procedure?

After you rinse the excess dye off the slide.

What ingredient is responsible for the difference between the physical consistency and the two media used?

Agar is in the slant which makes it a solid vs liquid which does not have a slant.

What breaks down Lipids?

An enzyme called Lipase

Repelled

Because cells have a net negative charge on their surface, acidic/anionic dyes are repelled by the surface of the cell

Why does basic dye attach to bacteria?

Because the basic dye has a positive charge and the bacteria have a negative charge. Opposing charges attract to each other, the basic dye will attach itself to the cells causing them to become colored while leaving the background of the slide transparent.

What effect would occur if you reversed the order of the stains and added safranin as the primary dye and crystal violet as the counterstain?

Both would be stained by the safranin. When iodine is added, safranin would be "set" in the positive. The decolorizer would wash out the safranin and then application of the crystal violet would stain the positive.

What breaks down Casein in Milk?

Casease (its the enzyme that makes milk white)

Effect of Temperature on Growth

Changes in temperature can slow reaction speeds down or cause proteins to denature

What breaks down DNA?

DNase (deoxyribonuclease)

What is the differential agent and indicator in Columbia CNA with 5% sheep blood agar?

Differential Agent: 5% Blood (gives cells a nice red, opaque color. Purpose is to identify cells capable of breaking down red blood cells. Indicator: hemolytic activity

DNA Agar (DNA Hydrolysis Test)

Differential Agent: DNA Identifies organisms that can produce deoxyribonuclease (DNase)

What is the differential agent and indicator in Bile Esculin Agar?

Differential Agent: Esculin Identifies streptococci and enterococci that hydrolyze esculin to esculetin Indicator: Ferric Citrate -Esculetin reacts with ferric citrate to form a dark color (Bile Esculin Agar helps us identify the presence of enterococci within a culture)

What is the differential agent and indicator in MacConkey Agar?

Differential Agent: Lactose Indicator: Neutral Red (acid causes a shift from colorless to red as the pH drops below 6.8) (as seen in 1 & 2)

What is the differential agent and indicator in Eosin Methylene Blue agar?

Differential Agent: Lactose and/or Sucrose Indicator: Eosin (red) / Methylene Blue

What is the differential agent and indicator in mannitol salt agar?

Differential Agent: Mannitol Indicator: Phenol Red

Starch Agar (Differential media) (Starch Hydrolysis Test)

Differential Agent: Starch Used to identify the presence of α-amylase and oligo-1,6-glucosidase

Tributyrin Oil Agar (TBA) (Lipid Hydrolysis Test)

Differential Agent: Tributyrin Oil Used to identify the presence of lipases

What are the differential agent and indicator in Hekoten Enteric Agar

Differential Agents: Lactose, Sucrose, and Salicin (these are dissacharides) Indicators: Bromthymol Blue and Acid Fuschin Secondary Differential Agent: Sodium Thiosulfate (sulfate source) Secondary Indicator: Ferric Ammonium Citrate (iron source)

Acid-Fast Stain

Differentiates acid-fast bacteria (pink) from non-acid-fast bacteria (blue) differentiates between those microbes that possess mycolic acid (wax) in their cell walls (mycobacterium)

endospore stain

Differentiates between those microbes that can sporulate (ie Clostridium and Bacillus)

How does MacConkey Agar differentiate microbes?

Identifies microbes that can ferment lactose and produce acid as a byproduct Acid causes a shift from colorless to red as the pH drops below 6.8 Indicates a possible coliform (causes UTIs if it gets outside the intestinal tract. E. coli can cause UTIs

How does Eosin methylene blue agar differentiate between microbes?

Identifies microbes that can ferment lactose and/or sucrose Acid will cause a color change by interacting with the dyes -Heavy acid = dark colonies -Light acid = pink colonies Fermentation means that the microbe is a possible coliform bacteria

How does manitol salt agar differentiate between microbes?

Identifies microbes that can ferment mannitol and produce acid. Acid production will cause a yellow color change as the pH of the media drops below 6.8 Indicative of Staphylococcus aureus

How does the tribuytin oil agar test work?

Identifies the presence of lipases that hydrolyze lipids or triglycerides for transport into cells. Converts the fatty acids into acetyl-CoA through Beta oxidation

Milk Agar (Casein Hydrolysis Test)

Indicator is the clearing of the "white" color produced by the milk protein, casein - A is the best microbe of casease producer -casein - predominate protein within milk

What is the chemical Indicator in the starch hydroliysis test?

Iodine is added to specimen after incubation, and presence of residual starch is indicated by a blue black/or dark brown color. Clearing around a colony indicates absence of starch. Therefore, a lack of color after the addition of the iodine indicates that starch is missing and must have occurred through the production of alpha-amylase by the microbe.

(HEA) What does the sodium thiosulfate provide the organism?

It provides the organism with a form of sulfur to allow it to carry out anaerobic respiration and to produce hydrogen sulfide (H2S) as a byproduct. The hydrogen sulfide then reacts with the iron, supplied by the ferric ammonium citrate, to produce a black precipitate in the colonies which indicates Salmonella sp (if present) or Shigella sp (if black is absent)

Disease caused by spirochete, for which the negative staining technique would be appropriate to aid in the microbes identification.

Lyme disease

Gram Stain of Micrococcus luteus (pink) and Escherichia coli (purple) from Mixed Culture Samples

Micrococcus luteus Morphology: Rod Color: pink Gram reaction: Gram-negative Escherichia coli Morphology: coccus Color: purple Gram reactions: gram-positive

What is the differential agent in Milk agar?

Milk

Gram Stain of Oral Bacteria

Morphology & Color: Rod (pink) /Coccus (purple) Arrangement: Staphylococci Gram Reaction: Gram + - Purple, Gram - is Pink

Micrococcus luteus (negative stain)

Morphology - Coccus, Dye: Nigrosin Gram Positive

Bacillus cereus (negative stain)

Morphology - rod, Dye - Nigrosin food poisoning

Acid-fast Stain of Mycobacterium smegmatis

Morphology: bacilli Color: pink Acid-fast bacteria is used because they retain the primary dye.

Aquaspirillum itersonii

Morphology: spiral (spirochette), dye: nigrosin gram negative

Endospore Stain of Clostridium botulinum showing vegetative cells and endospores

Motphology of Endospore: Bacillus Color: blue Morphology of Vegetative cell: bacillus Color of vegetative cell: pink

obligate aerobe growth

Organism only grows in the presence of Oxygen. Organism growth within the top of the FTM media

Basic (cationic) dyes

Positively charged dyes such as crystal violet, saffranin, methylene blue, carbolfuschion, and malachite green (bind with negatively charged cell)

Malachite green

Primary dye for Endospore stain

Fluid Thioglycollate Media (FTM) Organisms

Pseudomonas fluorescens - growth at top / obligate aerobes -Escherichia coli - growth throughout tube, except top - facultative anaerobe -Clostridium sporogenes - growth everywhere except top / obligate anaerobe

Gram staining procedure

Reagent (gram-positive 1) Crystal Violet - violet

What is the dye that turns pink when it is oxidized?

Resazurin, it allows you to see the diffusion of the oxygen in the media

Difference between rheostat and the diaphragm

Rheostate adjust the voltage of electricity entering the bulb. The diapharam (which is found in the condenser) is used to adjust the level of light being passed on to your specimen.

If a microbe causes a black precipitate to form on Hektoen enteric agar from H2S production, what could the microbe possibly by? If the black precipitate is absent, then what could it be?

Salmonella - black present Shigella - black is absent

If a microbe changes to pink or orange color on Hektoen enteric agar from disaccharide fermentation, what CANNOT the microbe be?

Salmonella or Shigella

Mannitol Salt Agar

Selective Agent: 7.5% NaCl Favors the growth of halo-tolerant Staphylococcus A: Halo-tolerant bacteria

Bile Esculin Agar (Bile Esculin (BESC) Test)

Selective Agent: Bile Salt Inhibits all Gram (+) bacteria EXCEPT enterococci and Group D streptococci

Hektoen Enteric Agar

Selective Agent: Bile Salts Favors growth of Gram (-)

Phenylethyl Alcohol Agar

Selective Agent: Phenylethyl Alcohol Favors the growth of Gram (+) by inhibiting the growth of gram (-) Organisms: Staphylococcus aureus - gram (+) Enterococcus faecalis - gram (+) Escherichia coli - gram (-)

Eosin Methylene Blue Agar (Selective and differential media)

Selective Agents (inhibitory agents) : Eosin (red dye) and Methylene Blue (blue dye) Favor growth of Gram (-)

MacConkey Agar (MAC)

Selective Agents: Bile Salts and Crystal Violet Favors growth of Gram (-)

Columbia CNA with 5% Sheep Blood Agar

Selective Agents: Colistin (antibiotic) and Nalidixic (antibiotic) Acid (CNA) Favors growth of Gram (+) - inhibiting the growth of gram (-). Useful in identifying the pathogenic: -Staphylococcus aureus - beta hemotolic hemolysis pattern - Complete degradation -Streptococcus mitis - alpha hemolysis pattern - incomplete degradation -Enterococcus faecalia - gamma hemolysis pattern - no degradation

Solid Inoculations

Slants - best inoculated with a loop and fishtail technique Deeps - best inoculated with a needle using a stab technique Agar Plates - best inoculated with a loop

What does the clearing around a colony in the Starch hydrolysis test mean?

The colony had used up the starch and was able to produce alpha amylase.

Nigrosin stain

The common name of the dye used to perform a negative stain on Aquaspirillum itersonii. Nigrosin was used to perform the negative stain because it contains negatively charged chromatophore that is electrostatically repelled by the negative surface of cells. This causes the dyes to pool in the background leaving the cell clear. It is also an important technique when the true shape of cells must be observed, such as when trying to identify a spirochete.

What is the significance of the 2 temperatures for incubation? 25 (room temperature) and 37 (body temp)

The experience compares incubation at room temperature and body temperature to determine if a bacteria responds differently inside or outside the body.

What is the indicator in DNA agar? How does it work?

The indicator is Methyl Green dye, that forms a complex with DNA to give a blue green appearance (methyl green binds itself to DNA) If it metabolizes the DNA, ie breaks it down, DNA will disappear- removes the dye and causes media to turn clear To determine whether an organism can produce DNase, look for a zone of clearing following incubation.

Why use a negative stain?

The negative stain is used to observe shape of the bacteria, such as: cocci, rod, spiral

How does the milk agar work?

Used to identify the presence of casease which hydrolyzes casein (the primary protein in milk) Casein (protein) -> produces amino acids (casease (protease) breaks down the protein)

Fluid Thioglycollate Medium (FTM)

Useful for studying aerotolerance of a given microbe Excellent for cultivating obligate anaerobes and microaerophiles An oxygen gradient is created within the media

Starch -> alpha-glucose

When you catabolize starch, you create glucose. When you catabolize glucose, you produce ATP. If you are going to metabolize starch, you will break it down into multiple uses - fermentation or cellular respiration

rheostat or illumination control

Your rheostat should always be at its highest setting when in use so that your bulb produces the whitest and brightest light possible.

Agar

a polysaccharide found in seaweed and commonly used to prepare solid culture media. It causes a liquid to become solid. it will remain solid until heated to 100 degrees C and it cannot be metabolized by any known clinically relevant microbe.

Sterile

absence of all life including spores

Thermophile

adapted to grow above 40 C human body denatures at this temp

Psychrotroph (some cause infections within human hosts)

adapted to grow between 0 C and 30 C ie, blue bell ice cream (listeria)

Mesophile

adapted to grow between 15 C and 45 C focus most on because it includes body temperature.

when conducting a dry culture

add water to your slide to ensure that you can easily spread your culture to create a smear. making it easier to view the shapes and cellular features.

Negative stain were not heat-fixed

because it would impact the shape of the cells. The slide should be placed in the biohazard sharps container.

colonies or colony forming unit

cluster or accumulation of single cells growing in a single location

Differential Media

contain chemical indicators along with additional nutrients that may or may not be utilized by some species of microbes - Metabolism of the nutrient creates byproducts that cause the indicator to change colors

Selective Media

contain one or more inhibitory agents designed to prevent the growth of some microbes while favoring the growth of others. Inhibits the growth of certain media by a chemical agent.

pure cultures

contain only a single species of microbe

mixed cultures

contain two or more species of microbes in a single culture

Extreme Thermophile

grow best above 80 C

Psychrophile (not important to use because we never get that cold)

grow only below 20 C (refrigerator and freezer)

Dip and Swirl Technique

liquid broths are best inoculated using an inoculation loop which holds the fluid much like a film across its opening using the dip and swirl technique.

aseptic technique

method used to make the environment, the worker, and the patient as germ-free as possible

acid dyes (anionic dyes)

negative staining technique, they carry a negative charge.

facultative anaerobe growth

organisms that can survive with and without oxygen

obligate anaerobe growth

organisms that cannot survive around Oxygen. Growth only happens throughout the tube and not on top

General Media

permit the growth of all organisms regardless of type or nutritional peculiarities

sepsis

presence of contamination and an environemnt that contains microbes or other debris that is potentially infectious or harmful to those that are unaware of its presence.

Carbolfuschin dye

primary dye for acid-fast staining

failure to add iodine in a gram stain will affect

probable decolorization of gram positives and no noticeable effects on gram negatives

Trypicase Soy Agar (TSA)

provides both the nutrients to support the growth of microbes and a platform upon which those microbes can grow. TSA is in the bottom of the plate, but is suspended over the lid to the petri dish. This is called inverted position and is the most common position for plates that contain media to be in so as to reduce possible contaimination.

Serratia marcescens

red at 25 degrees C white at 37 degrees C

What is the indicator of lipids in the Tribuytin Oil Agar?

the clearing of the cloudiness originally produced by the presence of oil in the agar. Organism on the left could not produce lipase, but the organism on the right could.

inoculation

the process of collecting bacteria from the culture tubes and then moving them into a new medium.

What breaks down Starch?

the production of alpha-amylase

inoculum

the sample being transferred or collected.

why do you hold the open culture tube at a 45 degree angle?

to minimize the culture's exposure to contamination, pass the mouth of the culture tube back and forth over the flame 3 or 4 times.

autoclaves

use heat and pressure to sterilize. 121 degree C at 15 psi for 30 minutes

simple stain dye / basic (cationic) technique

use of a single basic dye (crystal violet, methylene bue, carbolfuchsin or safranin)

Heat-fixed smear prep

used for simple and differential staining techniques.

streak plates

used to isolate microbes from a mixed culture

Staphylococcus epidermidis

yellow

Hemolytic Activity

β (beta) hemolysis: complete degradation α (alpha) hemolysis: incomplete degradation γ (gamma) hemolysis: no degradation