Histological and Hematological Analyses to Evaluate the Effects of Bacillus subtilis in Heat-Stressed Rats

Abstract

Source: Sorokulova, I., et al. Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain. J. Vis. Exp. (2016)

This video presents histological and blood microscopy analyses used to evaluate the protective effects of Bacillus subtilis in rats exposed to heat stress.

Protocol

1. Histological Analysis

1. Remove small intestine samples from each experimental rat by surgical procedure. Fix samples in Bouin’s Fixative, embed in paraffin, prepare 6 µm sections and stain sections with hematoxylin and eosin using standard procedures.
2. Use a high-resolution microscope system for measuring height of intestinal villi and total mucosal thickness in each sample (magnification 100X). Analyze 4 samples from each rat and make at least twenty measurements in each sample. Express results in micrometers.

2. High-Resolution Light Microscopy of Blood

1. Use a vibration isolation platform as a base for the microscope system and video camera and computer to record the live images. Calibrate test images using a Richardson slide.
2. Place 7 µl of freshly drawn blood from each rat on a glass slide, coverslip, photograph and record ten image frames of 72 × 53.3 µm² in each sample (magnification 1,700X).
3. Measure the concentration of vesicles using software that provides high-resolution direct-view optical images in real time. Examine a minimum of 20 image frames for each experimental condition.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate buffered saline (PBS)	Sigma-Aldrich, St. Louis, MO	P4417
Bouin’s Fixative	Electron Microscopy Sciences, Hatfield, PA, USA	15990
Richardson test slide	Electron Microscopy Science, Hatfield, PA, USA	80303
Sony DXC-33 Video camera	Sony
Light microscope optical system	CitoViva Technology Inc., Auburn,AL
Image-Pro Plus software	Media Cybernetics, MD, USA