Question

You are a researcher working for the USDA. After 2 years of effort, you have isolated in pure culture a new, highly virulent bacterium from duck feces that is responsible for several major outbreaks of deaths in mammalian wildlife from contaminated pond water in the South. Based on 16S rRNA sequence comparison, you have determined that this new bacterium is distantly related to the gram-negative bacterium V. cholerae, and you have named the new strain Vibrio birdsii. You have subsequently determined that V. birdsii is sensitive to tetracycline (i.e., it cannot grow in its presence) but is resistant to ampicillin. You obtained a Vibrio plasmid that you then used to construct a suitable E. coli shuttle vector for V. birdsii, which you could use for genetic manipulation in E. coli and then transfer into V. birdsii by transformation. You have also determined that guinea pigs, which have good innate immune systems, are an excellent animal model for the disease, which results primarily from uptake through drinking contaminated water, followed by colonization of the gut and bloody diarrhea, and then by invasion of intestinal cells with spreading to lymph nodes and spleen, and finally death by dehydration and/or organ failure. You are interested in identifying virulence factors associated with disease in guinea pigs. You have decided to use IVET as a strategy to identify these virulence factors. After infection and harvesting of the intestine, spleen, and lymph nodes of the guinea pigs, you have isolated a number of colonies on MacConkey agar plates from your initial screen: 10 white colonies, 15 pink colonies, and 75 red colonies.

How do you interpret the results from the initial screen? (Be sure to explain why there are three different colony colors.) What would you do with the pink colonies?

Answer 1

Answer-

According to the given question-

Based on the above information given in the paragraph above - a researcher  isolated the  pure culture of virulent bacterium from duck feces which causes several diseases and responsible for  deaths of wildlife mammalian who drink contaminated pond water . by using 16S rRNA sequence methodology the researcher identified it as a gram-negative bacterium which have close relation with V. cholerae and named it V. birdsii which are tetracycline sensitive but ampicillin resistant. Vibrio plasmid was constructed and manipulated in E. coli and again transfer into V. birdsii. this construct was tested in guinea pigs and the virulence factors was tested on growing colony on MacConkey agar plates which gives three color of 10 white colonies, 15 pink colonies, and 75 red colonies.  

• Vibrio cholerae are comma-shaped, belongs to the group of Gram-negative, facultative, anaerobic bacteria , responsible for causing diarrhea.
• MacConkey agar plates- It is a type of selective and differential culture medium used for the isolation and differentiation of Gram-negative bacteria. the composition of macConkey agar have Lactose , Bile, salts, Peptone, Sodium chloride , Crystal violet , Neutral red .and agar. the salt and Crystal violet prevent the growth of fastidious Gram-negative bacteria as well as the growth of Gram-positive bacteria but those negative bacteria that are capable of tolerating the salt concentration can grow in this media.
• MacConkey agar is also used for the selection of those gram negative bacteria which have the capability to ferment lactose present in the medium.
• the gram negative bacteria convert the lactose sugar into acid due to which the pH of medium decreases (below 6.8) that was observed by indicator neutral red dye and changes its color to pink. while those are not capable of fermenting the lactose sugar remains white or colorless.

according to the given question the three colonies are White , pink and red. the white colony are non lactose fermenting bacteria such as Proteus, Salmonella, and Shigella. Red or dark pink colony are fast lactose fermenting bacteria such as species of Escherichia, Klebsiella, and Enterobacter while the light pink color colony is due to slow lactose fermenting species of bacteria such as  Providencia, and Serratia,which after 24 hours become colorless.