Microbiology Lab 5-7

Triple Sugar Iron (TSI) Slant Test Color Identification

-Red = Alkaline environment (no fermentation) -Yellow = Acidic environment from fermentation → slant = Glucose Fermentation → butt = Sucrose and/or Lactose Fermentation -Broken agar = Gas production -Black = Hydrogen Sulfide Production

Culture media based on consistency

-Solid medium -Liquid medium -Semi-solid medium

The Four Quadrant Streak Technique

-Purpose: To obtain pure, isolated colonies -Principle: Spread a large amount of bacteria over the plate's surface area •The amount of bacteria is diluted until individual cells are spread on the surface of the plate. •Each individual cell grows into a single colony. •All of the cells in this colony are genetically identical. -Each species of bacteria produces a distinctive colony appearance: 1. Shape & Size (round, filamentous, irregular) 2. Edge (smooth or irregular) 3. Color 4. Opacity (transparent, translucent or iridescent) 5. Surface (smooth, irregular, dull, glossy)

TSI interpretation

Use this TSI Interpretation Sheet in order to narrow down/identify your Gram- Negative organism. Be sure to compare these results with those seen on the Enterotube.

Culture media

•Also called "growth media" •A liquid or gel designed to support the growth of microorganisms. •Contains nutrients needed to sustain a microbe. •Can vary in many ingredients: example: nutrients that microbes utilize for growth.

Semi-solid media

•Contains 0.5% agar •Ex: Motility medium

aseptic tips

•Work near the Bunsen burner •Use the flame to kill microorganisms on surfaces and to re-sterilize rims and lids •Minimize culture and container exposure to open air

BE + organisms

are able to grow in the presence of bile salts and will... -Hydrolyze Esculin in the medium resulting in the formation of glucose and a compound called Esculetin -Esculetin + Ferric Citrate → black diffusible complex

Enterotube test day 1: inoculate

insert image -Do not poke holes: Fermentation reactions use the broken needle to break the plastic seal

Mannitol Salt Agar (MSA)

selective for staphylococci

MacConkey (MAC)

selects for gram negative bacteria

Phenylethyl Alcohol (PEA)

selects for gram positive bacteria

Catalase

the enzyme that breaks H2O2 into H2O + O2 -Bubbling? Due to evolution of O2 gas!

Hydrogen Peroxide

used as a topical disinfectant in wounds. -H2O2 is a potent oxidizing agent that can wreck havoc on a cell! -Any cell that uses or can live in the presence of O2 must have a way to get rid of the peroxide . . . One of those ways is by producing the enzyme catalase.

Novobiocin Sensitivity Test

used in differentiation coagulase negative staphylococci

What is a Bacterial Culture?

•Bacteria have to be grown ("cultured") for them to be identified. •Using appropriate procedures, they have to be grown separately (isolated in colonies) on culture media and obtained as a pure isolate for further study.

Liquid media

•Does not contain agar •For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture. •Ex: Nutrient broth

Liquid cultures

•Inoculate by adding the cells with pipettes or syringes. •Uses: Blood culture, Sterility tests, Oxygen requirements; •Disadvantage: Does not provide a pure culture.

Undefined media

•Provide special nutrients •Contains complex ingredients *exact percent composition is unknown Ex: bovine serum albumin (BSA)

Defined/Synthetic media

•Provide special nutrients •Purest and most consistent cell culture environment •Entirely free of animal-derived components •Ex: peptone water (1% peptone + 0.5% NaCl in water)

lawn culture

•Provides a uniform surface growth of the bacterium •Prepared by flooding the surface of the plate with a liquid suspension of bacterium •Application example : Antibiotic sensitivity testing

Culture methods include

•Quadrant Streak •Lawn culture •Liquid culture

enriched media

•Type of special media •Substances like blood, serum, egg are added to the basal medium. •Used to grow bacteria that are exact in their nutritional needs. •Ex: Blood agar, Chocolate agar •Products are derived from sheep, bovine or human sources and contain complex mixes of albumins, lipids and other nutrients.

Simple/Basal media

•Unsupplemented Media •Promotes the growth of many types of microorganisms which do not require any special nutrient supplements •Ex: Nutrient Broth, Nutrient Agar

Selective media

•Used for the growth of only select microorganisms •An inhibitory substance is added to a solid media

Resistance to Neisseria Gonorrheae

•Vancomycin, which is able to kill most Gram-positive organisms, although some Gram-positive organisms such as Lactobacillus and Pediococcus are intrinsically resistant •Polymyxin, also known as colistin, which is added to kill most Gram-negative organisms except Neisseria, although some other Gram-negative organisms such as Legionella are also resistant •Nystatin, which can kill most fungi

TSI slant test contains

•a pH-sensitive dye (phenol red) •1% lactose •1% sucrose •0.1% glucose •sodium thiosulfate •ferrous sulfate or ferrous ammonium sulfate

Solid media

•contains 2% agar •Colony morphology, pigmentation, & hemolysis can be appreciated. •Ex: Nutrient agar, Blood agar

aseptic techniques

"Sterile" Techniques are used to avoid contamination of sterile media and equipment when working with live cell cultures and reagents and media that will be used for such cultures.

Blood Agar Plate (BAP)

(used in strep tests) contains bovine heart blood that becomes transparent in the presence of hemolytics.

Week 5 scenario

- Female 25 yr - Chronic UTI - Etiologic agent of UTI? Techniques: -Gram Stain -Culture: 4 Quadrant Streak MEDIA -Nutrient Agar -MacKonkey Agar

How to use the Enterotube Interpretation Sheet

-"Zero" = a completely negative result -"100" = a completely positive result -Values between 10 - 80 are generally unreliable for interpretation (since they represent partial positive/negative test results) and should only be considered, if at all, once all other compartments have been reviewed. -Begin by reviewing your results that are completely positive or negative. You can do this by using the "positive/negative" examples shown in the previous slide and by comparing it to an un-used demo tube located in J223. •Ex: Observe glucose → completely positive result → look for any organisms that have 100 → record a tally next to that organism. Repeat this for all compartments. -Review compartments that have partial positive and partial negative results. •Ex: An observed compartment is yellow but with a slightly orange tint compared to the demo. In this instance, if "yellow" is a negative result, you may consider looking for organisms with a ~ 0 - 30 results on the

Proper Storage and Labeling

-Agar plates are stored upside down to prevent contamination from condensation settling on the agar -All labeling is done on the bottom of the agar plate 1. Group Name/# 2. Date 3. BIO205 (Lab Section) 4. Bacteria (A/B? G+/G-)

Gram Positive Cocci: Catalase Negative

-All non-enterococcus species are BEA negative (not black) -All enterococcus species are BEA positive (black)

Culture media are based on

-Consistency -Constituents -Special factors

selective media examples

-MacConkey (MAC) -Mannitol salt agar (MSA) -Phenylethyl alcohol (PEA)

Culture media based on constituents (ingredients)

-simple/basal medium -complex medium -undefined medium -synthetic/defined medium

Enterotube test

-The Enterotube contains several compartments with various biochemical media and tests. -Used to identify G -, enteric (intestinal) organisms

Non-selective versus selective media

-The non-selective media on the left allows of the growth of several different bacterial species and is overgrown with bacteria (whitish lines). -The selective media plate on the right only allows the bacteria Neisseria gonorrhoeae, to grow (white dots).

Minimum inhibitory concentration (MIC)

-USE: Determine the minimum concentration of an antibiotic you can use for it to still be a successful inhibitor -Principle: •Inoculate a liquid medium with: (a) A specific concentration of antibiotics (b) An invisible but high number of microorganisms •Observe the mixture. See if it changes from clear to cloudy (turbulent). •Turbulent: Microorganisms have grown to high levels, and the test substance is not inhibitory to them at that particular dilution.

Special culture media

-enriched media -selective media -differential/indicator media

Catalase test procedure

1. Inoculate bacteria onto a microscope slide. 2. Add a drop of hydrogen peroxide onto the bacteria. 3. If bubbles are formed the organism is catalase positive.

Measure the zone of inhibition

Antibiotic Diffuses Into Agar To Elicit Effect on Bacterial Cells -After incubation of culture for 18 to 24 hours measure the zone of inhibition

Yes, because all colonies look alike.

Did this streak plate culture start as a pure culture. How can you tell?

MIC Interpretation:

For Gram Negative Bacilli -Ex: Escherichia coli

Coagulase test image

Gelling of the plasma remains in place even after inverting the tube

BEA contains

Selective components: -Bile salts (inhibits Gram-positive staphylococci) -Sodium Azide (inhibits Gram-negative bacteria) Differential components: -Esculin and peptone as nutrient sources -Ferric citrate as a color indicator.

Triple Sugar Iron (TSI) Slant Test

It is often used in the differential identification of enteric bacteria. -Semi solid media with a slant and butt. -Use inoculating needle!

Urease media

Media tests the ability of an organism to produce an exoenzyme, called urease -yellow= acidic -pink= basic

Coagulase test using rabbit plasma

Principle: Measures free coagulase. -[The free coagulase secreted by Staphylococcus aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.]

Enterotube test day 2

Record ALL Resuts then... -Add Kovac's Solution to #4 -Add 2 drops of KOH followed by 2 drops of alpha-naphthol to #9

True

SO......Starting with a mixed culture, pure isolation of different species may be obtained by streaking on four quadrant! 3rd and 4th Quadrants should have isolated colonies

How are culture media prepared

Step 1: Gather & mix the appropriate ingredients Step 2: Sterilization by autoclaving: 15-20 psi; 121°C; for 15-20 minutes Step 3: Use aseptic techniques to pour the plates

Differential/indicator media

Substances incorporated into the medium (nutrients or color indicators) enable visible distinction of a biochemical characteristic of the microorganism

Mannitol Salt Agar (MSA)

The high concentration of salt (7.5%) selects for members of the genus Staphylococcus, since they can tolerate high saline levels. -Also contains the sugar mannitol and the pH indicator phenol red

Culture

The process of growing a bacterial or other biological entity in an artificial medium. •for Isolation of PURE COLONIES •for Identification (Genus species)

Catalase Test

This test detects the production of catalase.

Kirby-Bauer Test example

Which antibiotics tested would you use to treat an infection caused by the test organism? -E, B, G, or D Which antibiotic is the bacteria the most susceptible to? -E

Streaking

a technique used to isolate a pure strain from a single species of microorganism, often bacteria. •Transfer samples of resulting pure, isolated colonies to a new microbiological culture. •This organism can be grown on a new plate in order to be identified, studied, or tested.

Scenario:

Your Unknown has the following reactions: 1.Glu+ 2.Gas + 3.Lys + 4.Orn +/- 5.Cit - 6.Ind + 7.Ure - 8.Sorb + 9.Arab + 10.Lac + 11.PA - 12.Ado - -QUESTION: What is the Probable Identification of your Unknown?

Bile Esculin Agar (BEA)

a selective and a differential medium -only do this test if your organism is Catalase negative

MacConkey Agar (MAC)

is differential for lactose fermentation -Lactose fermenters= Pink colonies -Non lactose fermenters= colorless colonies

Mannitol Salt Agar (MSA)

is differential for mannitol fermentation (No Fermentation --- Fermentation)