Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

Summary

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

Abstract

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.

First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.

The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.

The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.

This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Introduction

Cryptococcus neoformans is a pathogenic yeast found ubiquitously around the globe that is associated with human disease primarily in immunosuppressed populations. C. neoformans most notably accounts for a significant cause of total annual deaths in sub-Saharan Africa due to infectious disease¹. The major clinical manifestation of cryptococcal infection is meningoencephalitis, which follows invasion of the central nervous system by transport in infected macrophages (Trojan horse manner) or direct crossing of the blood-brain barrier. C. neoformans expresses several virulence factors including the ability to replicate at....

Protocol

1. Preparation of India Ink Slide

1. Pipette 10 µL of cryptococcal sample onto a slide. Any circular yeast strain will work but for this experiment H99 was the only strain used.
   NOTE: If the sample is directly from culture media, diluting 1:2 with PBS or water can help prevent the India Ink from clumping.
2. Pipette 2 µL of India Ink stain onto the sample and mix by physically pushing the pipette tip to the sample and moving in a circular motion until the India Ink appears evenly distributed.
3. Place a coverslip over the sample by holding down the left edge of the coverslip against the surface of the slide, then gently and even....

Results

Images are first obtained by microscopy of India Ink slides using a bright field microscope coupled with a camera (Figure 1A). It is important to have cells separated and in sufficiently low density not to overwhelm the field of view, as well as to use enough stain to create contrast between cells and background. As stated in the protocol, the exact number of cells for an optimal image will vary depending on the sample, microscope, and object.......

Discussion

The critical steps of this technique are preparing the India Ink slide and acquiring the microscope images. While the algorithm has been successfully tested with a variety of slide and image techniques the recommended protocol is described in this manuscript. The polysaccharide capsule is detected based on the exclusion of India Ink particles from the domain of the capsule as these particles are too large to penetrate the polysaccharide fibril network. India Ink exclusion results in a bright circle on top of a dark backg.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
India Ink	Becton, Dickinson and Co.	261194
Fisherbrand Superfrost Microscope Slides	Fisher Scientific	12-550-143	25x75x1
Fisherfinest Premium Cover Glass	Fisher Scientific	12-548-B	22x22-1
Sally Hansen HardasNails Xtreme Wear Nail Polish	Sally Hansen	N/A	109 invisible
SAB Media	Sigma	S3306
Cryptotoccus neoformans	ATCC	208821	H99 strain
Olympus AX70 Microscope	Olympus	AX70TRF	Discontinued ; Bright Field Microscope
Qimaging Retiga 1300	Qimaging	N/A	Discontinued ; Camera Microscope Attachment
MATLAB	MathWorks	N/A	Most recent version recommended
Python Programming Language	Python	N/A	Version 2 necessary ; 2.7 recommended
Microsoft Excel	Microsoft	N/A	Most recent version recommended
Phosphate Buffered Saline (PBS)	Sigma	P3813