TITLE: Conidial production from M. fijiensis mycelium
AUTHORS: Leticia Peraza, Cecilia Rodríguez, Dalia Zapata

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Continuous </span><span style = "font-style:italic;">in vitro</span><span> conidial production from </span><span style = "font-style:italic;">Mycosphaerella fijiensis</span><span> mycelium. A method for </span><span style = "font-style:italic;">in vitro</span><span> production of conidia is described. </span><span style = "font-style:italic;">M fijiensis</span><span> was grown on culture medium containing V8 juice. Mycelium was macerated until a fine suspension was produced; 2 mL was carefully spread on V8 sporulation medium. The Petri dishes were incubated for 6 days at 20 °C, under continuous cool-white fluorescent and black light. After the incubation period, 2.4 mL of 1% gelatin was added to each Petri dish, the conidia carefully dislodged with a camel hair brush. Conidial production was notably stimulated by brush harvesting mycelial cultures. After 6 days of culture the first harvest was made and the same batch of petri dishes were put in the same culture conditions to stimulate new conidial production. Significant cumulative conidial production was observed after the second harvest.</span></div></div>

[STEPS]
?. V8 juice
[per litre]
?. [V8 sporulation (V8S) medium]
Calcium carbonate
[per litre]
?. Add Agar-agar
[per litre]
?. Mix step 1 and 2 in 1 L of distiller water for 15 min
?. Measure pH and adjut to pH 6
?. Sterilize at 1 pressure atmosphere and 120° C for 15 min
?. Pour 25 mL of V8 sporulation medium into each Petri dish,
wait 3 days to see if any contamination was present then it can be used
?. [Inoculation of ground mycelium in V8 sporulation medium for conidia production]
Ground mycelium using a mortar and pistile in a laminal flow cabinetcut 1 cm2 of mycelium and put it into a mortaradd 2.5 mL of destilled sterile watergrind to a non-pasty liquid consistencypour 2.5 mL of the grind to each Petri dish containing V8 sporulation mediumthe ground mycelium is distributed making circles, until the entire Petri dish is full of suspensionlet the Petri dish for 20 min to dry, then seal with kleen pack
?. [Inoculation of ground mycelium in V8 sporulation medium for conidia production]
The Petri dishes were put in the folowing culture condition for sporulationRoom temperature 20 °C Continuous light with cool-white fluorescent and black light (General Electric 15W/18) emitting radiant energy in the near-ultraviolet or black light spectral rangeTime in this conditions: for the firs harvest 5 days for the second harvest 3 days (same batch of petri dishes).
?. [Harvest of conidia]
Harvest of conidia  in a laminal flow cabinetTake 2400 μL of grenetine (1%) and add it to the mycelium grown in the V8S mediumSweep gently with the camel bruch Tilt the petri dish and with the help of the micropipette wash the mycelium with the same suspension After harvesting put all the suspension in a Falcon 50 mL tube, the resulting suspension must be cloudy
?. [ Conidia count]
After the harvest take a sample for counting,  if necessary you can dilute the suspension 1:10Use the  Sedgwick-Rafter chamber to count the conidia (it could be the Neubauer chamber) using a The following formula is used to calculate the concentration of conidiacells or spores/mL= ΣN x 1000 mm3  x dilution factor                                  A x D x FWere:N=Conidia countedA= field area 1 (mm2)D= depth of field (1 mm)F= numbers of counted fields (25)                 3. Once the concentration of conidia is calculated, then the concentration of the original suspension is adjusted to 200 conidia per uL                 4. If the conidia are not going to be used immediately, they can be stored at 4 ° C