225 Lab Final (page 1)

Medium:  starch agar

Substrate: starch

Products: glucose, maltose, dextrins

Reagent: Lugol's iodine

Medium: skim milk agar

Substrate: casein

Products: amino acids, peptides 

Reagent: none used

Medium: DNase agar 

Substrate: ds DNA

Products: nucleotides 

Reagent: methyl green in agar (bound to DNA)

Medium: spirit blue agar 

Substrate: lipid (triglyceride)

Products: glycerol, fatty acids 

Reagent: spirit blue in agar (bound to triglycerides)

Amylase Test: + = colorless zone around organism after iodine is added

Caseinase Test: + = clear zone around organism

DNase Test: + = colorless zone around organism

Lipast Test: + = blue/clear zone around organisms, blue ppt. in organisms

B. subtilis produces both amylase and caseinase.

S. aureus produces DNase.  

Selective media allows one type of bacteria to grow while inhibiting the growth of other types.  

Differential media will change appearance based upon the type of bacteria growing in it.  

Media is often constructed so that both selective and differential properties are present.  The addition of certain chemicals produces a selective media in which the growth of certain bacteria is selectively inhibited.  While the addition of other chemicals produces a differential media in which obvious differences in appearance occur among the different growing bacteria (p. 55).

EMB agar and MacConkey's agar are both selective for gram negative bacteria (growth = gram negative) & differential for lactose fermentation

MSA is selective for staph and differential for mannitol fermentation.

• Medium: Eosin Methylene Blue (EMB) agar
• Substrate: Lactose
• Selective Ingredient: Eosin + Methylene Blue
• pH indicator: none listed

• Medium: MacConkey's agar
• Substrate: Lactose
• Selective Ingredient: Crystal Violet + bile salts
• pH indicator: Neutral red in agar

• Medium: mannitol salt agar
• Substrate: Mannitol
• Selective Ingredient: 7.5% NaCl
• pH indicator: Phenol red in agar

Only gram negative bacteria can grow in EMB agar.  

In EMB media, lactose fermenters are indicated by the incorporation of a dye from the media into the bacteria, thereby, turning the bacteria to a distinctly dark purple or shiny metallic color.  The result is often shown by E. coli but may also appear with other bacteria such as Klebsiella pneumoniae.  The colonies may show a distinctive arrangement of color within the colony, known as fisheye.  The center of the colony is a dark purple and the rest of the outer portion of the colony area is a lighter pink color.  The fisheye colonies are often produced by Enterobacter aerogenes. (from pg. 62)

Only gram negative bacteria can grow in MacConkey's agar.

Pink/red color to media/bacteria = lactose fermentation.  

• + organisms are
• E. coli
• and
• E. aerogenes

Growth = salt tolerant (staphylococci)

positive for mannitol fermentation = media is yellow

negative for mannitol fermentation = media remains pink in color

• Media: Tryptic Soy Broth (TSB)
• Substrate: Tryptophan
• Product: Indole
• Reagent: Kovac's layered on top
• pH indicator: none listed

• Media: MRVP broth
• Substrate: Glucose
• Products: Organic acids
• Reagents: Methyl red
• pH indicator: (Methyl red)

• Media: MRVP broth
• Substrate: Glucose
• Products: Non-acids
• Reagents: Barritts A and Barritts B
• pH indicator: none listed

• Media: Simmons' citrate agar
• Substrate: citrate
• Product(s): Na2CO3
• Reagents: none listed
• pH indicator: Bromthymol blue in agar

The utilization of the substrate tryptophan by the bacteria is indicated by testing for the presence of indole by adding Kovac's reagent to the 2-day broth culture.  If indole is present, the layer of Kovac's reagent will almost immediately turn red in color (p. 66)

E. coli produces a positive result like the one outlined above.

Methyl red is one of a group of molecules that are pH indicators whose color is dependent on the pH.  It is yellow in color under basic conditions and red in color under acidic conditions.  The uninoculated media is basic in pH and when methyl red is added, a yellowish color is observed.  If the glucose (substrate) has been fermented to mixed acids, a decidedly red color will appear (positive result).  p. 67

E. coli is an organism which will produce a positive result.  (red coloring throughout)

Positive Result = the whole tube will eventually turn pink.  This reaction is very slow.  

E. aerogenes produces the above result.  (whole tube eventually turns pink)

The bromthymol blue in the agar is green in color to start.  If the bacteria utilize citrate (the substrate), they will grow, produce alkaline end products, the pH will become basic, and the media/bromthymol blue will turn a royal blue color.  (p. 67)

E. aerogenes is a bacteria which will produce the above positive result.