Explaining spore counts, germination percentages
and CFU’s (colony forming units).
Spore count, germination percentage and colony forming units (CFUs) are measures that indicate the quantity and the viability of the active ingredient within a biological product to ensure that each batch meets the standards specified on the product’s label.
The spore count is a measure of quantity (number of spores), and the germination percentage is a measure of viability (percentage of alive spores). CFU is a measurement of the quantity of viable active ingredient.
In South Africa, Andermatt PHP products are currently registered with the spore count per gram provided on the label. However, each batch undergoes a germination percentage test as well as a CFU count in
order to ensure the correct quantity of viable active ingredient is present in each batch.
Spore count – Quantity of active
Spore count – Quantity of active Spores are individually counted at 400 times magnification. The guaranteed minimum for final product for Eco-Bb® is 2 x 109 (or two billion) spores per gram. The spore count cannot change over time, only the viability of each spore (whether it is living or dead).
Microbe Definition:
‘a living organism that on its own is too small to be seen without a microscope’
Germination – Viability of active
A germination test is done by suspending the final product in sterile water and growing it on enriched media. After 24 hours the spores are viewed at 400 times magnification and individual germinated spores are counted as a percentage of total spores counted (Fig. 1).
Figure 1: Spores of Beauveria bassiana shown with two spores germinating with germ tubes and ten ungerminated spores (x400 magnification).
Germination – Viability of active
CFU’s are quantified by suspending the final product in sterile water and growing it on enriched media. The growth of spores is left for 3-4 days until individual colonies are visible with the naked eye and can be counted (Fig. 2).
Figure 2: Colony forming units (CFU) as seen on agar.
Figure 1: Spores of Beauveria bassiana shown with two spores germinating with germ tubes and ten ungerminated spores (x400 magnification).
Eco-Bb® contains Beauveria bassiana. Reg. No. L8469, Act No. 36 of 1947. Andermatt PHP (Pty) Ltd, PO Box 207, Nottingham Road, 3280. Reg. No. 2003/007987/07.
FAQs
Is a product with a higher active ingredient concentration better than a product with a lower active ingredient concentration?
No – the concentration of a particular isolate in a product (the active ingredient used in different products) is dependent on the virulence of the isolate. Virulence is the harmfulness of an organism towards another (e.g. the harmfulness of Beauveria bassiana towards a red spider mite). The higher the virulence of the isolate, the lower the amount of isolate needed to kill its target. During product development, multiple strains are compared against each other for virulence and the dose required to kill its target. Once the product is formulated at a given concentration, it is tested in multiple field trials at various dose rates to determine the minimum effective field dose. Therefore, a registered product has been scientifically tested to ensure that the correct field dose rate for the product is indicated on the label.
For Eco-Bb®, why is the CFU/g lower than the spore count/g?
Eco-Bb® is formulated with oil so that multiple spores are encapsulated within each oil droplet. Therefore, when Eco-Bb® is placed on growth media, several spores together will grow into only 1 colony forming unit (see figure 3 below of spores in oil). An average CFU count for this product is 1 x 108 (hundred million) CFU per gram. Oil is used in the formulation to protect the spores from UV and desiccation, and to allow the spores to suspend in water when applied to the spray tank.
Why does spores/g appear on the Andermatt PHP product labels and not CFU/g?
At the time of registration, spores/g was the standard measure to indicate the concentration of the active ingredient in a fungal based product. Although germination percentage and CFU/g are not indicated on our labels, these tests are conducted on each batch to ensure that a high-quality product reaches our customers every time.
Why is spore count, germination and CFU important?
These measures provide quality control targets that production companies must meet to ensure that each batch of a particular product received by the customer is of the required standard.
Chantal Janks
Head of Research at Andermatt PHP
Spoortellings, ontkiemingspersentasies en “colony forming units” (CFU) (of kolonievormende eenhede (KVE) in Afrikaans) is maatstawwe wat die hoeveelheid en die lewensvatbaarheid van die aktiewe bestanddeel binne ʼn swam-gebasseerde biologies produk aandui om te verseker dat elke lot wat vervaardig word voldoen aan die standaarde wat op die produketiket gespesifiseer word.
Die spoortelling is ʼn maatstaf van hoeveelheid (aantal spore). Die ontkiemingspersentasie is ʼn maatstaf van lewensvatbaarheid (persentasie lewende spore). CFU meet die hoeveelheid lewensvatbare aktiewe bestanddeel.
In Suid-Afrika is Andermatt PHP-produkte tans geregistreer met die spoortelling per gram wat op die produketiket verskaf word. Elke lot ondergaan egter ʼn ontkiemingspersentasietoets en ontvang ʼn CFU- elling om te verseker dat die korrekte hoeveelheid lewensvatbare aktiewe bestanddeel in elke lot teenwoordig is.
Spoortelling – Hoeveelheid aktief
Spore word individueel getel by 400 maal vergroting. Die gewaarborgde minimum vir die finale produk vir Eco-Bb ® is 2 x 109 spore per gram. Die spoortelling kan nie oor tyd verander nie, slegs die lewensvatbaarheid van elke spoor (of dit lewend of dood is) kan verander.
Definisie van ‘n mikrobe:
”n lewende wese of organisme wat so klein is dat dit slegs deur n mikroskoop gesien kan word.’
Ontkieming – Lewensvatbaarheid van aktiefe
‘n Ontkiemingstoets word gedoen deur die finale produk in steriele water te suspendeer en dit op verrykte media te laat groei. Na 24 uur word die individuele ontkiemde spore en die totale spore onder 400 maal vergroting getel, Die individuele ontkiemde spore word uitgedruk as n persentasie van die totale getelde spore (Figuur 1).
CU – Hoeveelheid lewensvatbare aktiewe bestanddeel
CFU word gekwantifiseer deur die finale produk in steriele water te suspendeer en dit op verrykte media te laat groei. Die spore word vir 3-4 dae gelos om te groei totdat individuele kolonies met die blote oog sigbaar is en getel kan word (Figuur 2).
Figure 1: Spores of Beauveria bassiana-spore waarvan die twee spore met kiembuise reeds ontkiem het en tien spore wat nog nie ontkiem het nie (x400 vergroting).
Figuur 2: CFU“Colony forming units” soos gesien op agar plaatijies.
Algemene vrae
Is ʼn produk met ʼn hoër aktiewe bestanddeel konsentrasie beter as ʼn produk met ʼn laer aktiewe bestanddeel konsentrasie?
Nee – die konsentrasie van ʼn bepaalde isolaat in ʼn produk (die aktiewe bestanddeel wat in verskillende produkte gebruik word) is afhanklik van die virulensie van die isolaat. Virulensie is hoe skadelik een organisme teenoor ʼn ander is (bv. die skadelikheid van Beauveria bassiana teenoor ʼn rooispinmyt). Hoe hoër die virulensie van die isolaat, hoe laer is die hoeveelheid van die spesifieke isolaat wat nodig is om sy teiken dood te maak. Tydens produkontwikkeling word verskeie stamme met mekaar vergelyk vir virulensie en die dosis wat nodig is om sy teiken dood te maak. Sodra die produk teen ʼn gegewe konsentrasie geformuleer is, word dit in veelvuldige veldproewe teen verskeie dosisse getoets om die minimum effektiewe veld dosis te bepaal. Daarom is ʼn geregistreerde produk wetenskaplik getoets om te verseker dat die korrekte veld dosis vir die produk op die etiket aangedui word.
Rakende Eco-Bb®, waarom is die CFU/g laer as die spoortelling/g?
Eco-Bb® is geformuleer met olie sodat verskeie spore binne elke oliedruppel ingekapsuleer word. Dus, wanneer Eco-Bb® op groeimedia geplaas word, sal verskeie spore saam groei tot slegs 1 kolonievormende eenheid (sien figuur 3). ʼn Gemiddelde CFU-telling vir hierdie produk is 1 x 108 (honderd miljoen) CFU per gram. Olie word in die formulering gebruik om die spore teen UV en uitdroging te beskerm, en om die spore in water te laat suspendeer wanneer dit op die spuittenk toegedien word.
Waarom verskyn spore/g op Andermatt PHP produketiket en nie die CFU/g nie?
Ten tye van registrasie was spore/g die standaard maatstaf om die konsentrasie van die aktiewe bestanddeel in ʼn swamgebaseerde produk aan te dui. Alhoewel ontkiemingspersentasie en CFU/g nie op ons etikette aangedui word nie, word hierdie toetse op elke lot uitgevoer om te verseker dat ʼn produk van hoë gehalte ons kliënte elke keer bereik.
In die skets hier bo van ʼn biologiese produk, sal die resultaat van ʼn spoortelling 15 spore wees en die CFU-telling sal 7 CFU wees.
Waarom is spoortelling, ontkieming en CFU
belangrik?
Hierdie maatstawwe verskaf kwaliteitbeheer-teikens waaraan
produksiemaatskappye moet voldoen om te verseker dat elke produkreeks van ʼn spesifieke produk wat deur die kliënt ontvang word, van die vereiste standaard is.
Figuur 3: Beauveria bassiana-spore soos gesien onder ʼn mikroskoop in ʼn geformuleerde produk (x400 vergroting). Groepe spore word in oliedruppels ingekapsuleer. Een oliedruppel sal lei tot 1 CFU.
Chantal Janks
Hoof van navorsing by Andermatt PHP