TBL

Question 1

What is gram-negative bacteria

Answer 1

• bacteria that do not retain the crystal violet stain used in the gram-staining method => appears red/pink
• cell envelopes composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane

Question 2

V. natriegens is a halophile, what is halophile?

Answer 2

organism that thrives in or can tolerate saline conditions

Question 3

What are V. natriegens natural habitats

Answer 3

• brackish water
• estuarine mud

Question 4

what is brackish water

Answer 4

• water with salinity levels between seawater and freshwater
• may occur as estuaries or brackish fossil aquifers

Question 5

what is estuarine mud

Answer 5

the mud of the waters where seawater and freshwater meets

Question 6

V. natriegens are not photoluminescent, what are photoluminescent bacteria?

Answer 6

• light-producing bacteria
• predominantly present in sea water, marine sediments, the surface of decomposing fish and in the gut of marine animals
• free living or in symbiosis with animals: hosts provide safe home and nutrition. Hosts use the light produced by the bacteria for camouflage, prey and/or mate attraction
• luminescence reaction could also be for quorum sensing: ability to regulate gene expression in response to bacterial cell density

Question 7

Is V. natriegens free living, parasitic or mutualistic with other animals?

Answer 7

free living

Question 8

How do V. natriegens move?

Answer 8

by single polar flagellum

Question 9

What is a single polar flagellum

Answer 9

• single flagellum at one pole

Question 10

How is V. natriegens shaped?

Answer 10

comma-shaped vibrio

Question 11

What are the metabolic characteristics of Vibrio species?

Answer 11

facultatively anaerobic heterochemoorganotrophs

Question 12

what is a faculative anaerobe

Answer 12

can survive in the presence of oxygen, can use oxygen in aerobic respiration, but can also survive without oxygen via fermentation or anaerobic respiration.

Question 13

what is a heteroheterochemoorganotroph

Answer 13

• cannot produce its own food
• energy source: breaking down organic chemical compound
• obtain electrons or hydrogen from the organic compounds
• uses organic sources of carbon

Question 14

Are Vibrio species cyt-c oxidase positive or negative (TMPD test)? And what does that mean?

Answer 14

• positive => purple colour
• cyt-c oxidase positive organisms contain cytochrome c as part of their respiratory chain
• organisms lacking cytochrome c as part of their respiratory chain do not oxidise the reagent, leaving it colorless => negative

Question 15

Are Vibrio species catalase positive or negative in H₂O₂ test and what does that mean?

Answer 15

• usually catalase positive => bubbles when H₂O₂ added
• Test is used to identify organisms that produce catalase: detoxifies hydrogen peroxide by breaking it down into water and oxygen gas

Question 16

what do Vibro species do when under aerobc conditions

Answer 16

oxidise glucose fully to carbon dioxide using oxygen

Question 17

What do Vibrio species do under anaerobic conitions?

Answer 17

• respire using nitrate rather than oxygen as a terminal electron acceptor. Therefore they are nitrite positive (forms red precipitate) in sulfanilic acid / α-naphthylamine test because nitrate is reduced to nitrite
• most can also use glucose anaerobically by mixed acid fermentation, producing lactate, succinate, etc., but not usually ethanol, CO2 or H2. Therefore have a positive methyl red test

Question 18

what is methyl red test

Answer 18

• used to determine which fermentation pathway is used to utilize glucose
• In mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids)
• stable production of enough acid to overcome the phosphate buffer will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear => positive
• negative = yellow

Question 19

Vibrio species sources of nitrogen?

Answer 19

both organic and inorganic (NH⁴⁺, NO³⁻)

Question 20

what is a defined media

Answer 20

media that contain specific amounts of pure, chemically defined ingredients e.g. NaCl

Question 21

What is complex media

Answer 21

media that contain at least one ingredient that is a mixture of ill-defined components (peptides, sugars, vitamins, etc.) of unknown proportions

Question 22

in what conditions is the media autoclaved at?

Answer 22

121°C for 15 min at 15 psi (103 kPa)

Question 23

what is autoclaving

Answer 23

usesing steam under pressure to kill harmful bacteria, viruses, fungi, and spores on items that are placed inside a pressure vessel

Question 24

why does autoclaving have to be at 121°C and not just boiling temperature?

Answer 24

bacterial spores can survive 100°C

Question 25

Why does the pressure in autoclave have to be raised to 15 psi

Answer 25

so that the water doesn't boil until it reaches a temperature high enough to kill bacterial endospores

Question 26

*V. natriegens* is non-fastidious heterotroph, what does that mean?

Answer 26

organism that **does not require** special nutrients to be present in their culture medium to grow

Question 27

what does the defined media of non-fastidious heterotroph often contain?

Answer 27

* **phosphate**: pH buffer and phosphorus source * **salt**: regulate ionic strength * **carbon source** (usually a sugar, but amino acids can also be used) * **nitrogen source** (e.g. ammonium, nitrate, or glutamate) * various **trace elements** (Mg, Ca, Fe, Cu, etc.)

Question 28

what do fastidious microbes additionally need on top of the usual components of non-fastidious defined media

Answer 28

additional trace elements (Fe, Zn, Co, Mo, Cu, B, *etc*.), vitamins, amino acids, *etc*.

Question 29

what is the preferred complex medium for *V. natriegens*

Answer 29

* BHI (brain-heart infusion) broth * **BHI** is made by combining an **infusion** from boiled bovine or porcine **heart** and **brain** with a variety of other nutrients * 37 g·L−1 brain-heart infusion * 20 g·L−1 NaCl

Question 30

what is M9Na

Answer 30

A modified version (for halophilic *V. natriegens*) of the common defined medium for non-fastidious heterotrophs M9