Oxygen Requirements:  Fluid 

Thioglycolate Media
A                             B                                C                                      D                                E   


Organisms grown in Fluid Thioglycollate Media, a reducing media that uses sodium thioglycolate to chemically combine with dissolved oxygen to deplete the oxygen in the media. The top portion of the tube contains oxygen while the bottom of the tube is lacking molecular oxygen.  All organism except for Neisseria sicca were grown for 48 hours at 37o C.Neisseria sicca was grown for 72 hours at 37o C for best results.  Note: Both N. sicca and C. butrycium were grow in supplemented Thioglycollate Media.  A calcium chip can be seen in the bottom of the tube for N. sicca which is used as a buffer.  This media is aslo supplemented with vitamin K.   Note:  I buy prepared thioglycolate media and many times the media is "jostled" in shipping.  I will autoclave the tubes to drive off the oxygen from the bottom of the tubes if necessary.  The tube can be autoclave once after initial preparation without affecting the media.  Remember that for this media the lids on the tubes Must be tightened down! 
 
 (A)  Escherichia coli:  Facultative Anaerobe/Aerotolorent, grows both aerobically and anerobically, growth is seen throughout the tube but mainly in the middle to lower portion of the tube possibly preferring a slightly lower oxygen content.  Some are capable of growth respiring with oxygen and anaerobically by fermentation.   

 B) Neisseria sicca: Microaerophile, requires oxygen but at concentrations below atmosphere, grows just below the surface of the media but not at the top.  This organism is fastdidous and can take a  week or longer to see good growth.

(C) Clostridium butrycium: Obligate Anaerobe:  incapable of growth in the presence of oxygen, growth is seen approximatly 1/4 to 1/2 of the way from the top of the tube.   This organism can be hard to grow as well.  I start these cultures 2 weeks before needed and once i have an active culture it stay's in the incubator, kept warm, indefinatly.  If I inoculate the thioglycolate media and do not see growth, active bubble's rising from the bottom of the tube, within 48 hrs then I take a hot loop (inoculating loop that is red hot from flaming) and plunge it into the tubed media 3-5 times.  Care must be taken when doing this because of "splatter" and should only be done in a BSL II cabinet.  Point tube away from user when doing this.  

(D) Pseudomonas aeroginosa: Obligate Aerobe:  oxygen is required for growth, growth at the top of the tube only.  Organism will "settle" and sink into the media if longer than 24 hrs so for best results look at tube within the 24 hr time period.

(E) Staphylococcus aureus: Facultative Anaerobe/Aerotolerant, grows both aerobically and anerobically, growth is seen throughout the tube.  


      A                                                B                                                     C              


(A) Escherichia coli & (B) Staphylococcus aureus outer two tubes are Facultative Anaerobes/Aerotolorent, both grow aerobically and anerobically and can growth with or without molecular oxygen. The organism is capable of making ATP through respiration if molecualr oxygen is present but can switch to fermentation or anerobic respiration if molecular oxygen is absent. Growth is seen throughout, compared to the center tube, the tube but mainly in the middle to lower portion of the tube possibly preferring a slightly lower oxygen content  (B) : Clostridium butrycium: Obligate Anaerobe: incapable of making ATP through respiration and will die in the in the presence of oxygen, growth is seen approximatly 1/4 to 1/2 of the way from the top of the tube.


Neisseria sicca is a Microaerophile, requires oxygen because they can not ferment or do enerobic respiration but they need reduced  concentrations of molecuar oxygen, below atmosphere (between 1-10% oxygen), where high oxygen concetrations above that can be toxic to the cell.  Grows just below the surface of the media where oxygen concentrations are reduced but not at the top.  Note: for Neisseria sicca supplemented thioglycolate media is used which contains a calcium chip seen in the bottom of the tube. 
  
        Pseudomonas  aeruginosa was grown for 48 hours at 37oC.  Pseudomonas aeruginosa : Obligate Aerobe:  oxygen is required for growth,  growth at the top of the tube only where oxygen concetratins are the highest.  Note:Organism will "settle" and sink into the media if longer than 24 hrs.





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