Autoclave glass tips

[Karol]

I'm using 95%. My wife (who has a M.Sc in Medicinal Chemistry) tells me that's enough. Is she wrong?

Depends on whether it needs to be sterile or just sanitized. 95% does not have the same lethality as 70%.

 

[Beefriendly]

There are various degrees of sterilisation...sanitising is bad word to use as simply implies getting rid of....which can be anything from germs to whole populations of people.
To over simplify...3 major components to remove when sterilising anything....living bacteri, yeasts and spores.
70% Alchohol is universally accepted (and used throughout the scientific world) as being sufficient for killing off the vast majority of bacteria/fungi yeast etc.
But it's not good enough to kill many dormant bacterial spores...they require either dry or wet heat. Wet heat is considered via pressure cooker or autoclave as 15lb for at least 15 minutes. Dry heat is accepted as 180C for a 2 hours. They destroy/inactivate the proteins in the spores by different methods hence the temperature differences.
Both work...I prefer ovens as easier to to use than pressure cookers and no residual moisture left in sterilised item.

 

[B+.]

There are various degrees of sterilisation...sanitising is bad word to use as simply implies getting rid of....which can be anything from germs to whole populations of people.
To over simplify...3 major components to remove when sterilising anything....living bacteri, yeasts and spores.
70% Alchohol is universally accepted (and used throughout the scientific world) as being sufficient for killing off the vast majority of bacteria/fungi yeast etc.
But it's not good enough to kill many dormant bacterial spores...they require either dry or wet heat. Wet heat is considered via pressure cooker or autoclave as 15lb for at least 15 minutes. Dry heat is accepted as 180C for a 2 hours. They destroy/inactivate the proteins in the spores by different methods hence the temperature differences.
Both work...I prefer ovens as easier to to use than pressure cookers and no residual moisture left in sterilised item.

Lets be realistic. The tip is used to skim sperm off the mucous layer of the drones endophallus then inject it into the median oviduct of the queen. It is possible to contaminate the tip by touching the exoskeleton of the drone/queen or even by touching it with contaminated hands. I clean my hands with ethanol before starting an insemination so I believe that I am minimizing the risk of contamination from my hands....but..as soon as you touch the queen, you're contaminated again.
I do have a pressure cooker which is dedicated to cleaning II kit but Beefriendly's point about residual moisture is relevant. How does this compare with the moisture left by passing ethanol, then distilled water through it? I don't know but probably about the same(?). I found this (https://www.compliancenaturally.com...eVeSLQIRKNfZ6t3UCSiYR7FfjbGUZC4_azVz1u1dtNyjg ) that talks about why 70% ethanol is used in Pharma/Food facilities. Perhaps I should go back to using the pressure cooker but I'm still worried about whether the leur connector would disintegrate after going through a pressure cooker a few times. I'm sorry Beefriendly, but, I'm sure they'd not survive the oven even once.

I suppose this all boils down to the question: how clean is the queens median oviduct? Any comments?

 

[Beefriendly]

I'm sorry Beefriendly, but, I'm sure they'd not survive the oven even once.

Lets be realistic...I'm not taking a personal interest in the sterilisation of individual bits of your II kit. I'me explaining how sterilisation works. If you want more ideas on how to sterilise plastic bits of your II kit then UV light is good, but needs to be a strong source..
But it is far simpler to use 70% ethanol...as I've already explained.

 

[B+.]

But it is far simpler to use 70% ethanol...

In case anyone is wondering, regrettably, you can't drink it

 

[Beefriendly]

You can drink 70% ethanol. It's the same stuff that's in your current glass of vino/scotch G&T etc But more concentrated
You may be confusing it with methanol./iso propanol etc...... which are not good to drink...
Made some great "punches" in the past with bottles of 95% ethanol ..excise duty paid.

 

[B+.]

You can drink 70% ethanol. It's the same stuff that's in your current glass of vino/scotch G&T etc But more concentrated
You may be confusing it with methanol./iso propanol etc...... which are not good to drink...
Made some great "punches" in the past with bottles of 95% ethanol ..excise duty paid.

I meant ...that its denatured so people can't drink it

 

[Beefriendly]

I meant ...that its denatured so people can't drink it

NO. TOTAL RUBBISH..70% ethanol ,is not "denatured" it's the same alcohol that is in your current poison....and requires excise duty paid on it. I used to be in charge if our bonded storage area for the stuff. Even though it was used for science you couldn't avoid the excise duty.

You are mixing up methylated spirits with pure ethanol...Meths is adulterated with other substances to make it poisonous.

 

[Karol]

<snip>

I'm still worried about whether the leur connector would disintegrate after going through a pressure cooker a few times.

<snip>


I suppose this all boils down to the question: how clean is the queens median oviduct? Any comments?

If you are concerned about the plastic withstanding dry heat you might consider microwaving as a means of sterilization.

As for any implications of microbial contamination of the median oviduct then I suggest this would depend on the infecting organism. I can't imagine that accidental insemination with honeybee pathogens would be healthy. So for example does Nosema naturally get into the oviduct and if so what are the effects?

When it comes to sterile/aseptic processes then two wrongs don't make a right, i.e. just because it might be difficult to avoid contamination during handling doesn't IMHO excuse avoiding contamination from other sources.

 

[beebopper]

NO. TOTAL RUBBISH..70% ethanol ,is not "denatured" it's the same alcohol that is in your current poison....and requires excise duty paid on it. I used to be in charge if our bonded storage area for the stuff. Even though it was used for science you couldn't avoid the excise duty.

You are mixing up methylated spirits with pure ethanol...Meths is adulterated with other substances to make it poisonous.

Denatured is used in this context. It is a different use of word to proteins denatured.

Goggle it and looks at wiki. Do know how to do link with iPad.

 

[B+.]

Thank you for your input on alternative methods but I have successfully inseminated queens for quite some years following my wifes advice (she usually knows what she is talking about). Other people may be able to make use of it but there seems to be difficulties in using these alternatives that only come to light with practical experience of the equipment itself.

 

[maddydog]

Thank you for your input on alternative methods but I have successfully inseminated queens for quite some years following my wifes advice (she usually knows what she is talking about). Other people may be able to make use of it but there seems to be difficulties in using these alternatives that only come to light with practical experience of the equipment itself.

I guess this is the rub over the rhetoric. How many queens have you infected using your technique?

OP probably wishing he'd never asked.

 

[Karol]

In which case sanitization is all that you appear to need for the process.

One final little tip (no pun intended) going down the sanitizing route - a contact time of at least two minutes with 70% ethanol is recommended. If you use 70% ethanol there's no need for rinsing with water - just ensure that all the 70% ethanol has evaporated off to dryness. That way you not only ensure proper contact time, i.e. that all vegetative microbes have been killed, but also avoid potential contamination from the water rinse.

 

[mazzamazda]

Probably stupid questions as I am a complete newbie to all of this. Can all sanitizing be done with alcohol? Is 95% better than 70% as for some reason it is cheaper here at €0.75c for 250ml and is the contact time reduced with this concentration?

 

[B+.]

In which case sanitization is all that you appear to need for the process.

One final little tip (no pun intended) going down the sanitizing route - a contact time of at least two minutes with 70% ethanol is recommended. If you use 70% ethanol there's no need for rinsing with water - just ensure that all the 70% ethanol has evaporated off to dryness. That way you not only ensure proper contact time, i.e. that all vegetative microbes have been killed, but also avoid potential contamination from the water rinse.

Thank you for your input. I will take it under advisement (i.e. discuss it with Mrs B+) and, if necessary, change the ethanol but I am loathe to change a system that works unnecessarily.
I suspect that the internal diameter of the tip, micropipette and tubing (the tip measures 0.15mm at the business end) will delay evaporation of the ethanol in the way you describe, but, I can try it and see.

 

[B+.]

Probably stupid questions as I am a complete newbie to all of this. Can all sanitizing be done with alcohol? Is 95% better than 70% as for some reason it is cheaper here at €0.75c for 250ml and is the contact time reduced with this concentration?

The problem I attempted to convey earlier is that the material that some of the parts is made of doesn't react well to alcohol exposure. You don't know that until you try it (or someone tells you) though. The same may be true of the other methods discussed here.

 

[Karol]

Thank you for your input. I will take it under advisement (i.e. discuss it with Mrs B+) and, if necessary, change the ethanol but I am loathe to change a system that works unnecessarily.
I suspect that the internal diameter of the tip, micropipette and tubing (the tip measures 0.15mm at the business end) will delay evaporation of the ethanol in the way you describe, but, I can try it and see.

I understand your concern regarding the capillary action of the micropipette retarding evaporation but you will have the same problem with a water rinse the difference being that there is a risk of growth with water (not so with 70% ethanol) and 70% ethanol is more volatile so will dry out better than distilled water. So long as you check the tips are dry before use there should be no problem. Obviously any residues of ethanol will be harmful to harvesting so the check is critical.

 

[B+.]

I understand your concern regarding the capillary action of the micropipette retarding evaporation but you will have the same problem with a water rinse the difference being that there is a risk of growth with water (not so with 70% ethanol) and 70% ethanol is more volatile so will dry out better than distilled water. So long as you check the tips are dry before use there should be no problem. Obviously any residues of ethanol will be harmful to harvesting so the check is critical.

The distilled water is simply to flush the harmful effects of the ethanol out of the system before introducing filtered saline. It's the saline that acts as a "hydraulic fluid" in the syringe. It doesn't actually come into contact with the sperm as there is an air-space between them. The objective is to get a solid column of drone sperm in the micro-pipette which can be used subsequently or posted to other breeders.

One of the other issues is that the tip must be kept moist (with filtered saline) when collecting drone sperm. An air-space is kept between the sperm column and the saline, but this must be expelled before making contact with a drop of the sperm from the stack and the sperm from the new drone. Then, capillary action is used to skim the sperm off the surface of a mucous bed. It's not easy to hold the drone steady while this happens and the tip can become plugged with mucous. That part is a pain!

 

[beebopper]

but I am loathe to change a system that works unnecessarily.

Well why would you.
As usual masses of over complication here which corresponds to 'know it alls' interference.

KISS

 

[B+.]

Well why would you.
As usual masses of over complication here which corresponds to 'know it alls' interference.

KISS

Indeed. I would make changes if there was a benefit/simplification but the changes suggested seem to bring their own difficulties with them.
It seems to be six-of-one, half-a-dozen of the other. The process is difficult/complicated enough without further things that could go wrong. It's amazing that bees manage it, when you come to think of it!