Lab 8

Continuation of Neurospora crassa genetics

Purpose

The objectives of this laboratory exercise on October 17, 2012, were to 1) pick perithecia from Neurospora crassa crosses initiated during Lab 5 on September 26, 2012, and squash them to observe asci and ascospores, and 2) observe field-collected mushrooms.

Materials and Methods 

N. crassa SMRP10 x CSP-1-GFP
Field-collected mushrooms
Microscope slides
Cover slips
Dissecting needle
Dropper bottle with water 
Spray bottle with 70% ethanol
Paper towels
Metal striker
Bunsen burner
Canon PowerShot SD550 digital camera
Olympus SZ30 zoom stereo microscope 
Olympus CX31 compound microscope 

A. N. crassa cross 

While observing a N. crassa SMRP10 x CSP-1-GFP cross under a dissecting microscope, and using a sterile technique (described in my blog for Lab 2 on September 5, 2012), a dissecting needle was used to pick several mature perithecia. The perithecia were placed in a drop of water on a microscope slide. A cover slip was applied and then gently pressed with the handle of the dissecting needle to squash the perithecia and release asci containing ascospores. Fungal structures were observed using a dissecting microscope and a compound microscope that had been set to Köhler according to instructions also received during Lab 2. Photographs were taken through the eyepiece of the microscope using a digital camera. Ascospores with nuclei expressing green fluorescent protein (GFP) also were observed using fluorescence microscopy.

B. Field-collected mushrooms 

A number of field-collected mushrooms that are part of a Department of Plant Pathology and Microbiology collection at Texas A&M University were made available for macroscopic observation. 

Results

A. N. crassa cross

Three-week-old N. crassa SMRP10 x CSP-1-GFP cross used to pick perithecia.

Mature perithecia that were ready to shoot ascospores, as observed using a dissecting microscope (4X). Photograph cropped and enlarged to show detail.

A group of four football-shaped ascospores that were shot from perithecia onto the lid of the culture dish, as observed using a dissecting microscope (4X). Photograph cropped and enlarged to show detail.

Two squashed perithecia released asci containing ascospores, as observed using a compound microscope (10X). Photograph cropped and enlarged to show detail. Each ascus contained eight ascospores.

Using fluorescence microscopy, multiple nuclei expressing GFP could be observed in four of the eight ascospores in this ascus from the N. crassa SMRP10 x CSP-1-GFP cross. Photograph cropped and enlarged to show detail. Nuclei in all eight ascospores should have expressed GFP. It is not known why the nuclei in only half of the ascospores expressed GFP.

B. Field-collected mushrooms