MY Bubbles & More Tests as Usual

MY Bubbles & More Tests as Usual 

Class: Tuesday, October 21, 2013

FINALLY.  Bacteria J had it's first reaction today!  Last week we made a streak plate of our bacteria (shared the plate with another group...theirs is yellow) so that this week we could test how it reacted to a bath of hydrogen peroxide.  A reaction indicates the presence of the enzyme catalase, which breaks down the H2O2.  Usually the reaction will occur in aerobic, aerotolerant, or facultative anaerobic bacteria .  

Bacteria streak before reaction. Bacteria J is
the orange one on the left

Jess adding the hydrogen peroxide 

Woohoo! We have a positive test result.  It may
look like the bubbles are coming from the other
bacteria, but they really are from Bacteria J! Also note how
Bacteria J seems to be dissipating as the reaction occurs....interesting

MY bubbles.  

Test 1:

      Culturing Bacteria J in an anaerobic environment.  The culture medium is broth that has thioglycollate added to it.  The thioglycollate is a reducing agent which dissolves some of the oxygen in the tube so that there are oxygenated and deoxygenated areas for the bacteria to possibly grow in.  The location of growth indicates the bacteria's oxygen requirements.  

Here, we can see the differentiation in oxygen distribution that the thioglycollate produced.  There is a small section of pink broth at the top while the remainder is clear.  Oxygen is only present in the pink section and absent in the rest of the culture medium.  The results of this test will indicate Bacteria J's true oxygen requirements.  

The rest of the tests we did today are both selective  and differential.  Selective mediums favor the growth of one type of bacteria over another.  Differential mediums distinguish between a few different types of bacteria.  The results will look different depending on the bacteria present 

Test 2:

      Eosin Methylene Blue (EMB) Agar.  This growth test is used to isolate gram-negative bacteria that ferment lactose or sucrose.  EMB is a selective medium because the eosin and methylene blue dyes inhibit the growth of gram-postive bacteria and it is a differential medium because the lactose and sucrose sugars it contains definitively distinguishes between bacteria that do ferment sucrose/lactose and those that don't.  There are a few different outcomes here that we could see in growth and color:

1. No bacterial growth = gram-positive bacteria
2. Dark blue colony w/ green metallic sheen = significant fermentation of sucrose and/or lactose
3. Pink colonies= some fermentation
4. Colorless (but still present) colonies = no fermentation

Sierra inoculated the EMB agar plate in the pattern F U S.  Because we're cool like that, ya know? 

Sierra, expert inoculator.  

Test 3:

      Blood Agar Plate.  Another differential and selective media.  Blood agar (from sheep's blood) is highly enriched with nutrients and thus helps isolate fastidious bacteria that have trouble growing else.  I bet Bacteria J is like this, the fussy little thing.  Because blood agar contains well, blood cells, it differentiates bacteria that completely lyse RBCs (beta-hemolytic), partially lyse RBCs (alpha-hemolytic), and do not lyse RBCs (non-hemolytic).

Test 4:
      Phenylethyl Alcohol (PEA).  PEA selectively inhibits gram-negative bacterial growth because it interferes with DNA synthesis.  Preeety simple.

Look at that focus! 

Test 5:
      Mannitol Salt Agar.
      Selectivity: salt tolerance; bacteria must be able to tolerate a 7.5% NaCl concentration
      Differentiation: mannitol fermentation; acid from mannitol fermentation changes the pH indicator in the medium (phenol red) from red to yellow.  If the bacteria can ferment mannitol, a yellow zone will appear around the colony.  Pathogenic bacteria have this mannitol fermentation ability, so let's hope we see some yellow! (yes, we actually want a pathogenic bacteria....it's exciting).

      

Test 6:
      MacConkey Agar. MacConkey Agar selects for gram-negative bacteria.  The crystal violet and bile salts in the medium prohibit the growth of gram-positive bacteria.  The presence of lactose in the medium differentiates between lactose fermenting and non-lactose fermenting bacteria.  This is indicated by the color of the colonies; lactose-users turn pink-red.  non-lactose fermenters remain colorless.

Can I get an F U S??

Test 7 (last one, I promise!) 

      DNase hydrolysis test.  To determine bacteria's ability to digest DNA, which they could do if they secrete DNase enzyme.  Pathogenic bacteria use DNase to clog up infected tissues so they can move more easily from cell to cell.  We will be adding HCl to the plate in the next lab period to truly investigate the DNase production of Bacteria J.  

Does it eat DNA? Only time shall tell....
Also, we shared a plate again with Mary and Jess
They're pretty neat.  We like 'em.  

Now time for some lab group lovin'. 

Matt, you know you love us.