Streptomyces/O.D. reading

Yang, Doo Suck microbe at DONALD.HANHYO.CO.KR
Sun Dec 8 18:50:45 EST 1996

-- [ From: Yang, Doo Suck * EMC.Ver #2.5.02 ] --

Strepcomyces is a genus included in Actinomycetes.
It grows in mycelia, like fungi. So it's difficult, even impossible to
OD. Usually Streptomyces growth is measured by Dried Cell Weight (DCW) 
or Packed mycelial Volume (PMV). 

There are three ways to make sample equally suspended. One thing is using
mild homoginization, which makes fragments of mycelium.

The other method is using enzyme which breaks cell wall, like lysozyme. That
makes protoplast. 

The third is using medium on which Streptomyces makes spores.
Then use spores to prepare equally suspended samples.
See below for medium composition.

Sporulation medium (R2YE, Hopwood et al., 1985)
	Sucrose	                     		       103	g
	K2SO4                   		          0.25	g
	MgCl2¡¤6H2O     	              	        10.12	g
	Glucose                    			        10	g
	Casamono acids              		          0.1	g
	Yeast extract 	                  		  5	g
	Agar                     		      		 22	g
	Distilled water                  			800	ml

	After autoclaving add following constituent;
	KH2PO4 (0.5%)                   		 10	ml
	CaCl2¡¤2H2O (3.68%)              		 80	ml
	L-proline (20%)                	  		 15	ml
	TES buffer (5.73%, pH 7.2)       		100	ml
	NaOH (1N)                       			  5	ml
	Trace element solution*            		  2	ml
	*Trace element solution (per liter)
	ZnCl2                            			 40	mg
	FeCl3¡¤6H2O  		                 	       200	mg
	CuCl2¡¤2H2O                      		 10	mg
	MnCl2¡¤4H2O                    			 10	mg
	Na2B4O7¡¤10H2O                   		 10	mg

-------- REPLY, Original message follows --------

> Date: Thursday, 05-Dec-96 03:19 PM
> From: aj mccardell             \ Internet:    (mccardaj at esvax.dnet.dupont.
> To:   microbio at net.bio.net     \ Internet:    (microbio at net.bio.net)
> Subject: Streptomyces/O.D. reading
> I am trying to develop a sample preparation procedure with Streptomyces
and am
> having some difficulty.  Our standard sample prep procedure is to use a 
> plastic stick to pick colonies from an agar plate.  Since the Streptomyces
I am
> growing are quite 'crusty' and grow into the agar somewhat, I can't
> them from the agar surface in this way.  I thought I would switch to
> them up in broth and then adjust the O.D. to my target range.  The problem
> that I can't vortex to achieve a uniform suspension of cells.  These
things are
> like big strings that don't disperse.  Also, when I have dug colonies from
> plates, I haven't been able to vortex the sample into any kind of
> I am currently using BHI media to grow these samples.  They are growing
> alright.  I was thinking about switching to Bennett's medium in the hopes
> getting a little bit more luxurious growth.  Does anyone have any
> for media?  How do people take O.D. readings of Streptomyces?  (I've tried
> incubating plates at higher humidity and have been thinking about
ammending the
> media with a little Tween but that's the limit of my less-than-brilliant
> ideas.)
> Hope someone can help.
> Thanks,
> A.J.

-------- REPLY, End of original message --------

Hanhyo Institutes of Tehchnology
Fermentation Lab. 
461-6 Chunmin-dong, Yoosung-ku,
Taejun, 305-390, KOREA

Tel. 82-42-866-9136
Fax. 82-42-866-9129

Research Scientist

Yang, Doo Suck

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